Teresa E. Pawlowska

Organization of genetic variation in individuals of arbuscular mycorrhizal fungi

Arbuscular mycorrhizal (AM) fungi (Glomeromycota) are thought to be the oldest group of asexual multicellular organisms. They colonize the roots of most land plants, where they facilitate mineral uptake from the soil in exchange for plant-assimilated carbon. Cells of AM fungi contain hundreds of nuclei. Unusual polymorphism of ribosomal DNA observed in individual spores of AM fungi inspired a hypothesis that heterokaryosis—that is, the coexistence of many dissimilar nuclei in cells—occurs throughout the AM fungal life history. Here we report a genetic approach to test the hypothesis of heterokaryosis in AM fungi. Our study of the transmission of polymorphic genetic markers in natural isolates of Glomus etunicatum, coupled with direct amplification of rDNA from microdissected nuclei by polymerase chain reaction, supports the alternative hypothesis of homokaryosis, in which nuclei populating AM fungal individuals are genetically uniform. Intrasporal rDNA polymorphism contained in each nucleus signals a relaxation of concerted evolution, a recombination-driven process that is responsible for homogenizing rDNA repeats. Polyploid organization of glomeromycotan genomes could accommodate intranuclear rDNA polymorphism and buffer these apparently asexual organisms against the effects of accumulating mutations.

The mycorrhizal status of plants colonizing a calamine spoil mound in southern Poland

Abstract The arbuscular mycorrhizal (AM) status of two plant communities on a calamine spoil mound (rich in cadmium, lead and zinc) in southern Poland was surveyed: an undisturbed grassland community and an early succession community that developed after complete removal of the surface layer of the calamine substrate about 10 years earlier. The undisturbed site harbored 40 herbaceous species making up 87% of the absolute cover. AM colonization was recorded in 25 species accounting for 77% of the relative cover. Species with 51–75% AM root colonization such as Festuca ovina and Leontodon hispidus dominated the undisturbed turf, contributing 45% to the relative cover. Carex ssp. were the most abundant nonmycorrhizal plants and accounted for 9% of the relative cover. Spores of Glomus aggregatum, G. constrictum, G. fasciculatum, G. pansihalos, Glomus sp. and Entrophospora sp. averaged 25 per 100 g dry substrate at the undisturbed site. The disturbed site was colonized by 25 species accounting for 17% of the absolute cover. Among the AM plants, most abundant were the species with up to 20% AM root colonization, such as Agrostis stolonifera and Thymus pulegioides, wich accounted for 24% of the relative cover. Nonmycorrhizal species, such as Biscutella laevigata,Cardaminopsis arenosa, Gypsophila fastigiata and Silene vulgaris, dominated the early succession community and contributed 64% to the relative cover. Spores of G. fasciculatum and Entrophospora sp. averaged 20 per 100 g dry substrate at the disturbed site.

Heavy-metal stress and developmental patterns of arbuscular mycorrhizal fungi.

ABSTRACT The rate of global deposition of Cd, Pb, and Zn has decreased over the past few decades, but heavy metals already in the soil may be mobilized by local and global changes in soil conditions and exert toxic effects on soil microorganisms. We examined in vitro effects of Cd, Pb, and Zn on critical life stages in metal-sensitive ecotypes of arbuscular mycorrhizal (AM) fungi, including spore germination, presymbiotic hyphal extension, presymbiotic sporulation, symbiotic extraradical mycelium expansion, and symbiotic sporulation. Despite long-term culturing under the same low-metal conditions, two species, Glomus etunicatum and Glomus intraradices, had different levels of sensitivity to metal stress. G. etunicatum was more sensitive to all three metals than was G. intraradices. A unique response of increased presymbiotic hyphal extension occurred in G. intraradices exposed to Cd and Pb. Presymbiotic hyphae of G. intraradices formed presymbiotic spores, whose initiation was more affected by heavy metals than was presymbiotic hyphal extension. In G. intraradices grown in compartmentalized habitats with only a portion of the extraradical mycelium exposed to metal stress, inhibitory effects of elevated metal concentrations on symbiotic mycelial expansion and symbiotic sporulation were limited to the metal-enriched compartment. Symbiotic sporulation was more sensitive to metal exposure than symbiotic mycelium expansion. Patterns exhibited by G. intraradices spore germination, presymbiotic hyphal extension, symbiotic extraradical mycelium expansion, and sporulation under elevated metal concentrations suggest that AM fungi may be able to survive in heavy metal-contaminated environments by using a metal avoidance strategy.

Effects of Metal Phytoextraction Practices on the Indigenous Community of Arbuscular Mycorrhizal Fungi at a Metal-Contaminated Landfill

ABSTRACT Phytoextraction involves use of plants to remove toxic metals from soil. We examined the effects of phytoextraction practices with three plant species (Silene vulgaris, Thlaspi caerulescens, and Zea mays) and a factorial variation of soil amendments (either an ammonium or nitrate source of nitrogen and the presence or absence of an elemental sulfur supplement) on arbuscular mycorrhizal (AM) fungi (Glomales, Zygomycetes) at a moderately metal-contaminated landfill located in St. Paul, Minn. Specifically, we tested whether the applied treatments affected the density of glomalean spores and AM root colonization in maize. Glomalean fungi from the landfill were grouped into two morphotypes characterized by either light-colored spores (LCS) or dark-colored spores (DCS). Dominant species of the LCS morphotype were Glomus mosseae and an unidentified Glomus sp., whereas the DCS morphotype was dominated by Glomus constrictum. The density of spores of the LCS morphotype from the phytoremediated area was lower than the density of these spores in the untreated landfill soil. Within the experimental area, spore density of the LCS morphotype in the rhizosphere of mycorrhizal maize was significantly higher than in rhizospheres of nonmycorrhizal S. vulgaris or T. caerulescens. Sulfur supplement increased vesicular root colonization in maize and exerted a negative effect on spore density in maize rhizosphere. We conclude that phytoextraction practices, e.g., the choice of plant species and soil amendments, may have a great impact on the quantity and species composition of glomalean propagules as well as on mycorrhiza functioning during long-term metal-remediation treatments.

Detection of a novel intracellular microbiome hosted in arbuscular mycorrhizal fungi

Arbuscular mycorrhizal fungi (AMF) are important members of the plant microbiome. They are obligate biotrophs that colonize the roots of most land plants and enhance host nutrient acquisition. Many AMF themselves harbor endobacteria in their hyphae and spores. Two types of endobacteria are known in Glomeromycota: rod-shaped Gram-negative Candidatus Glomeribacter gigasporarum, CaGg, limited in distribution to members of the Gigasporaceae family, and coccoid Mollicutes-related endobacteria, Mre, widely distributed across different lineages of AMF. The goal of the present study is to investigate the patterns of distribution and coexistence of the two endosymbionts, CaGg and Mre, in spore samples of several strains of Gigaspora margarita. Based on previous observations, we hypothesized that some AMF could host populations of both endobacteria. To test this hypothesis, we performed an extensive investigation of both endosymbionts in G. margarita spores sampled from Cameroonian soils as well as in the Japanese G. margarita MAFF520054 isolate using different approaches (molecular phylotyping, electron microscopy, fluorescence in situ hybridization and quantitative real-time PCR). We found that a single AMF host can harbour both types of endobacteria, with Mre population being more abundant, variable and prone to recombination than the CaGg one. Both endosymbionts seem to retain their genetic and lifestyle peculiarities regardless of whether they colonize the host alone or together. These findings show for the first time that fungi support an intracellular bacterial microbiome, in which distinct types of endobacteria coexist in a single cell.

Microdissection of Shoot Meristem Functional Domains

The shoot apical meristem (SAM) maintains a pool of indeterminate cells within the SAM proper, while lateral organs are initiated from the SAM periphery. Laser microdissection–microarray technology was used to compare transcriptional profiles within these SAM domains to identify novel maize genes that function during leaf development. Nine hundred and sixty-two differentially expressed maize genes were detected; control genes known to be upregulated in the initiating leaf (P0/P1) or in the SAM proper verified the precision of the microdissections. Genes involved in cell division/growth, cell wall biosynthesis, chromatin remodeling, RNA binding, and translation are especially upregulated in initiating leaves, whereas genes functioning during protein fate and DNA repair are more abundant in the SAM proper. In situ hybridization analyses confirmed the expression patterns of six previously uncharacterized maize genes upregulated in the P0/P1. P0/P1-upregulated genes that were also shown to be downregulated in leaf-arrested shoots treated with an auxin transport inhibitor are especially implicated to function during early events in maize leaf initiation. Reverse genetic analyses of asceapen1 (asc1), a maize D4-cyclin gene upregulated in the P0/P1, revealed novel leaf phenotypes, less genetic redundancy, and expanded D4-CYCLIN function during maize shoot development as compared to Arabidopsis. These analyses generated a unique SAM domain-specific database that provides new insight into SAM function and a useful platform for reverse genetic analyses of shoot development in maize.

In vitro propagation and life cycle of the arbuscular mycorrhizal fungus Glomus etunicatum

Progress in understanding the biology of arbuscular mycorrhizal fungi is hampered by the limited number of species that can be successfully propagated and studied in vitro . We report the establishment of monoxenic cultures of Glomus etunicatum in association with excised Ri T-DNA transformed carrot roots. The fungus can be propagated in vitro using monoxenically formed resting spores and/or colonized root fragments. Modified Whites medium buffered with 10 mM MES (pH 6) or MOPSO (pH 6.5) was most optimal for the host root growth as well as for G. etunicatum spore germination and mycorrhiza formation. The number of resting spores formed in vitro correlated positively with the length of roots occupied by arbuscular mycorrhizal structures, including arbuscules and vesicles. Spores first appeared in dual cultures within two weeks of root inoculation. Sporulation was asynchronous and continued until root senescence. Under applied culture conditions, spores achieved mature appearance within 5–7 d after their initiation. Approximately 6% of monoxenic spores were aborted at different stages of their development. Although G. etunicatum spores formed in vitro exhibited general morphological and anatomical similarity to soil-borne inoculum, they were significantly smaller and had thicker spore walls than their soil-borne counterparts. Caution should, therefore, be exercised in utilizing the in vitro system as a model of growth and development of glomalean fungi in soil.

Clonality and Recombination in the Life History of an Asexual Arbuscular Mycorrhizal Fungus

Arbuscular mycorrhizal (AM) fungi in the phylum Glomeromycota colonize roots of the majority of land plants and assist them in the uptake of mineral nutrients in exchange for plant-assimilated carbon. In the absence of sexual reproductive structures and with asexual spore morphology conserved since the Ordovician, Glomeromycota may be one of the oldest eukaryotic lineages that rely predominantly on asexual reproduction for gene transmission. Clonal population structure detected in the majority of AM fungi examined to date supports this hypothesis. However, evidence of recombination found in few local populations suggests that genetic exchanges may be more common in these organisms than is currently recognized. To explore the significance of clonal expansion versus genetic recombination in the life history of modern Glomeromycota, we examined the global population of a cosmopolitan fungus Glomus etunicatum and made inferences about the population structure and the occurrence of recombination in the history of this species. We sampled eight loci from 84 isolates. We found that although the global population of G. etunicatum showed a pattern of significant differentiation, several haplotypes had a broad geographic distribution spanning multiple continents. Molecular variation among the sampled isolates indicated an overwhelmingly clonal population structure and suggested that clonal expansion plays an important role in the ecological success of modern Glomeromycota. In contrast, a pattern of homoplasy consistent with a history of recombination suggested that gene exchanges are not completely absent from the life history of these organisms, although they are likely to be very rare.

Multinucleate Spores Contribute to Evolutionary Longevity of Asexual Glomeromycota

Arbuscular mycorrhizal fungi (Glomeromycota) are the dominant symbionts of land plants and one of the oldest multicellular lineages that exist without evidence of sexual reproduction. The mechanisms that protect these organisms from extinction due to accumulation of deleterious mutations in the absence of sexual recombination are unclear. Glomeromycota reproduce by spores containing hundreds of nuclei, which represents a departure from the typical eukaryotic developmental pattern, where a multicellular organism is re‐created from a uninucleate propagule. To understand whether the multinucleate spore makeup may have contributed to the evolutionary success of Glomeromycota, we examined the dynamics of spore nuclei in Glomus etunicatum using live three‐dimensional imaging and mathematical models. We show that the spores are populated by an influx of a stream of nuclei from the surrounding mycelium rather than by divisions of a single founder nucleus. We present evidence that mechanisms of selection are likely to operate at the level of individual nuclei. On the basis of mathematical analyses of the effects that these nuclear dynamics have on the population mutation load, we postulate that the developmental patterns of sporogenesis have adaptive significance for moderating the accumulation of deleterious mutations and may have contributed to the evolutionary longevity of Glomeromycota.

Minimal genomes of mycoplasma-related endobacteria are plastic and contain host-derived genes for sustained life within Glomeromycota

Significance Arbuscular mycorrhizal fungi (AMF) are soil fungi associated with the majority of land plants worldwide. They supply plants with mineral nutrients in exchange for photosynthates. Most AMF harbor endobacteria from the Mollicutes class. Exploring metagenomes of endobacterial populations in three AMF species, we discovered that these endobacteria have minimal genomes and are metabolically dependent on their fungal host. Despite vertical transmission, endobacterial genomes are uniquely plastic. In addition, the endobacteria contain multiple genes horizontally transferred from fungi. Many of these genes encode products thought to interact with fungal host proteins. Overall, the endobacterial genomes reveal a tightly knit network of interactions with the fungal host and highlight the importance of associations between bacteria and fungi. Arbuscular mycorrhizal fungi (AMF, Glomeromycota) colonize roots of the majority of terrestrial plants. They provide essential minerals to their plant hosts and receive photosynthates in return. All major lineages of AMF harbor endobacteria classified as Mollicutes, and known as mycoplasma-related endobacteria (MRE). Except for their substantial intrahost genetic diversity and ability to transmit vertically, virtually nothing is known about the life history of these endobacteria. To understand MRE biology, we sequenced metagenomes of three MRE populations, each associated with divergent AMF hosts. We found that each AMF species harbored a genetically distinct group of MRE. Despite vertical transmission, all MRE populations showed extensive chromosomal rearrangements, which we attributed to genetic recombination, activity of mobile elements, and a history of plectroviral invasion. The MRE genomes are characterized by a highly reduced gene content, indicating metabolic dependence on the fungal host, with the mechanism of energy production remaining unclear. Several MRE genes encode proteins with domains involved in protein–protein interactions with eukaryotic hosts. In addition, the MRE genomes harbor genes horizontally acquired from AMF. Some of these genes encode small ubiquitin-like modifier (SUMO) proteases specific to the SUMOylation systems of eukaryotes, which MRE likely use to manipulate their fungal host. The extent of MRE genome plasticity and reduction, along with the large number of horizontally acquired host genes, suggests a high degree of adaptation to the fungal host. These features, together with the ubiquity of the MRE–Glomeromycota associations, emphasize the significance of MRE in the biology of Glomeromycota.