A TAL effector-like protein of an endofungal bacterium increases the stress tolerance and alters the transcriptome of the host.

Symbioses of bacteria with fungi have only recently been described and are poorly understood. In the symbiosis of Mycetohabitans (formerly Burkholderia) rhizoxinica with the fungus Rhizopus microsporus, bacterial type III (T3) secretion is known to be essential. Proteins resembling T3-secreted transcription activator-like (TAL) effectors of plant pathogenic bacteria are encoded in the three sequenced Mycetohabitans spp. genomes. TAL effectors nuclear-localize in plants, where they bind and activate genes important in disease. The Burkholderia TAL-like (Btl) proteins bind DNA but lack the N- and C-terminal regions, in which TAL effectors harbor their T3 and nuclear localization signals, and activation domain. We characterized a Btl protein, Btl19-13, and found that, despite the structural differences, it can be T3-secreted and can nuclear-localize. A btl19-13 gene knockout did not prevent the bacterium from infecting the fungus, but the fungus became less tolerant to cell membrane stress. Btl19-13 did not alter transcription in a plant-based reporter assay, but 15 R. microsporus genes were differentially expressed in comparisons both of the fungus infected with the wild-type bacterium vs. the mutant and with the mutant vs. a complemented strain. Southern blotting revealed btl genes in 14 diverse Mycetohabitans isolates. However, banding patterns and available sequences suggest variation, and the btl19-13 phenotype could not be rescued by a btl gene from a different strain. Our findings support the conclusion that Btl proteins are effectors that act on host DNA and play important but varied or possibly host genotype-specific roles in the M. rhizoxinica-R. microsporus symbiosis.

Lipid metabolic changes in an early divergent fungus govern the establishment of a mutualistic symbiosis with endobacteria.

The recent accumulation of newly discovered fungal-bacterial mutualisms challenges the paradigm that fungi and bacteria are natural antagonists. To understand the mechanisms that govern the establishment and maintenance over evolutionary time of mutualisms between fungi and bacteria, we studied a symbiosis of the fungus Rhizopus microsporus (Mucoromycotina) and its Burkholderia endobacteria. We found that nonhost R. microsporus, as well as other mucoralean fungi, interact antagonistically with endobacteria derived from the host and are not invaded by them. Comparison of gene expression profiles of host and nonhost fungi during interaction with endobacteria revealed dramatic changes in expression of lipid metabolic genes in the host. Analysis of the host lipidome confirmed that symbiosis establishment was accompanied by specific changes in the fungal lipid profile. Diacylglycerol kinase (DGK) activity was important for these lipid metabolic changes, as its inhibition altered the fungal lipid profile and caused a shift in the host-bacterial interaction into an antagonism. We conclude that adjustments in host lipid metabolism during symbiosis establishment, mediated by DGKs, are required for the mutualistic outcome of the Rhizopus-Burkholderia symbiosis. In addition, the neutral and phospholipid profiles of R. microsporus provide important insights into lipid metabolism in an understudied group of oleaginous Mucoromycotina. Lastly, our study revealed that the DGKs involved in the symbiosis form a previously uncharacterized clade of DGK domain proteins.

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

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.

Nondegenerative Evolution in Ancient Heritable Bacterial Endosymbionts of Fungi.

Bacterial endosymbionts are critical to the existence of many eukaryotes. Among them, vertically transmitted endobacteria are uniquely typified by reduced genomes and molecular evolution rate acceleration relative to free-living taxa. These patterns are attributable to genetic drift-dominated degenerative processes associated with reproductive dependence on the host. The degenerative evolution scenario is well supported in endobacteria with strict vertical transmission, such as essential mutualists of insects. In contrast, heritable endosymbionts that are nonessential to their hosts and engage occasionally in horizontal transmission are expected to display deviations from the degenerative evolution model. To explore evolution patterns in such nonessential endobacteria, we focused on Candidatus Glomeribacter gigasporarum ancient heritable mutualists of fungi. Using a collection of genomes, we estimated in Glomeribacter mutation rate at 2.03 × 10(-9) substitutions per site per year and effective population size at 1.44 × 10(8) Both fall within the range of values observed in free-living bacteria. To assess the ability of Glomeribacter to purge slightly deleterious mutations, we examined genome-wide dN/dS values and distribution patterns. We found that these dN/dS profiles cluster Glomeribacter with free-living bacteria as well as with other nonessential endosymbionts, while distinguishing it from essential heritable mutualists of insects. Finally, our evolutionary simulations revealed that the molecular evolution rate acceleration in Glomeribacter is caused by limited recombination in a largely clonal population rather than by increased fixation of slightly deleterious mutations. Based on these patterns, we propose that genome evolution in Glomeribacter is nondegenerative and exemplifies a departure from the model of degenerative evolution in heritable endosymbionts.

'Candidatus Moeniiplasma glomeromycotorum', an endobacterium of arbuscular mycorrhizal fungi.

Arbuscular mycorrhizal fungi (AMF, subphylum Glomeromycotina) are symbionts of most terrestrial plants. They commonly harbour endobacteria of a largely unknown biology, referred to as MRE (Mollicutes/mycoplasma-related endobacteria). Here, we propose to accommodate MRE in the novel genus 'Candidatus Moeniiplasma.' Phylogeny reconstructions based on the 16S rRNA gene sequences cluster 'Ca.Moeniiplasma' with representatives of the class Mollicutes, whereas phylogenies derived from amino acid sequences of 19 genes indicate that it is a discrete lineage sharing ancestry with the members of the family Mycoplasmataceae. Cells of 'Ca.Moeniiplasma' reside directly in the host cytoplasm and have not yet been cultivated. They are coccoid, ~500 nm in diameter, with an electron-dense layer outside the plasma membrane. However, the draft genomes of 'Ca.Moeniiplasma' suggest that this structure is not a Gram-positive cell wall. The evolution of 'Ca.Moeniiplasma' appears to be driven by an ultrarapid rate of mutation accumulation related to the loss of DNA repair mechanisms. Moreover, molecular evolution patterns suggest that, in addition to vertical transmission, 'Ca.Moeniiplasma' is able to transmit horizontally among distinct Glomeromycotina host lineages and exchange genes. On the basis of these unique lifestyle features, the new species 'Candidatus Moeniiplasma glomeromycotorum' is proposed.

Molecular evolution patterns reveal life history features of mycoplasma-related endobacteria associated with arbuscular mycorrhizal fungi.

The mycoplasma-related endobacteria (MRE), representing a recently discovered lineage of Mollicutes, are widely distributed across arbuscular mycorrhizal fungi (AMF, Glomeromycota). AMF colonize roots of most terrestrial plants and improve plant mineral nutrient uptake in return for plant-assimilated carbon. The role of MRE in the biology of their fungal hosts is unknown. To start characterizing this association, we assessed partitioning of MRE genetic diversity within AMF individuals and across the AMF phylogeographic range. We further used molecular evolution patterns to make inferences about MRE codivergence with AMF, their lifestyle and antiquity of the Glomeromycota-MRE association. While we did not detect differentiation between MRE derived from different continents, high levels of diversity were apparent in MRE populations within AMF host individuals. MRE exhibited significant codiversification with AMF over ecological time and the absence of codivergence over evolutionary time. Moreover, genetic recombination was evident in MRE. These patterns indicate that, while MRE transmission is predominantly vertical, their complex intrahost populations are likely generated by horizontal transmission and recombination. Based on predictions of evolutionary theory, we interpreted these observations as a suggestion that MRE may be antagonists of AMF. Finally, we detected a marginally significant signature of codivergence of MRE with Glomeromycota and the Endogone lineage of Mucoromycotina, implying that the symbiosis between MRE and fungi may predate the divergence between these two groups of fungi.

Symbioses between fungi and bacteria: from mechanisms to impacts on biodiversity.

Symbiotic interactions between fungi and bacteria range from positive to negative. They are ubiquitous in free-living as well as host-associated microbial communities worldwide. Yet, the impact of fungal-bacterial symbioses on the organization and dynamics of microbial communities is uncertain. There are two reasons for this uncertainty: (1) knowledge gaps in the understanding of the genetic mechanisms underpinning fungal-bacterial symbioses and (2) prevailing interpretations of ecological theory that favor antagonistic interactions as drivers stabilizing biological communities despite the existence of models emphasizing contributions of positive interactions. This review synthesizes information on fungal-bacterial symbioses common in the free-living microbial communities of the soil as well as in host-associated polymicrobial biofilms. The interdomain partnerships are considered in the context of the relevant community ecology models, which are discussed critically.

Sequencing the Genomes of the First Terrestrial Fungal Lineages: What Have We Learned?

The first genome sequenced of a eukaryotic organism was for Saccharomyces cerevisiae, as reported in 1996, but it was more than 10 years before any of the zygomycete fungi, which are the early-diverging terrestrial fungi currently placed in the phyla Mucoromycota and Zoopagomycota, were sequenced. The genome for Rhizopus delemar was completed in 2008; currently, more than 1000 zygomycete genomes have been sequenced. Genomic data from these early-diverging terrestrial fungi revealed deep phylogenetic separation of the two major clades-primarily plant-associated saprotrophic and mycorrhizal Mucoromycota versus the primarily mycoparasitic or animal-associated parasites and commensals in the Zoopagomycota. Genomic studies provide many valuable insights into how these fungi evolved in response to the challenges of living on land, including adaptations to sensing light and gravity, development of hyphal growth, and co-existence with the first terrestrial plants. Genome sequence data have facilitated studies of genome architecture, including a history of genome duplications and horizontal gene transfer events, distribution and organization of mating type loci, rDNA genes and transposable elements, methylation processes, and genes useful for various industrial applications. Pathogenicity genes and specialized secondary metabolites have also been detected in soil saprobes and pathogenic fungi. Novel endosymbiotic bacteria and viruses have been discovered during several zygomycete genome projects. Overall, genomic information has helped to resolve a plethora of research questions, from the placement of zygomycetes on the evolutionary tree of life and in natural ecosystems, to the applied biotechnological and medical questions.

Arbuscular mycorrhizal colonization of giant sequoia (Sequoiadendron giganteum) in response to restoration practices.

Interactions with soil microbiota determine the success of restoring plants to their native habitats. The goal of our study was to understand the effects of restoration practices on interactions of giant sequoia Sequoiadendron giganteum with arbuscular mycorrhizal (AM) fungi (Glomeromycota). Natural regeneration of Sequoiadendron is threatened by the absence of severe fires that create forest canopy gaps. Generating artificial canopy gaps offers an alternative tool for giant sequoia restoration. We investigated the effect of regeneration practices, including (i) sapling location within gaps, (ii) gap size and (iii) soil substrate, on AM fungal colonization of giant sequoia sapling roots in a native giant sequoia grove of the Sierra Nevada, California. We found that the extent of AM fungal root colonization was positively correlated with sapling height and light availability, which were related to the location of the sapling within the gap and the gap size. While colonization frequency by arbuscules in saplings on ash substrate was higher relative to saplings in mineral soil, the total AM fungal root colonization was similar between the substrates. A negative correlation between root colonization by Glomeromycota and non-AM fungal species indicated antagonistic interactions between different classes of root-associated fungi. Using DNA genotyping, we identified six AM fungal taxa representing genera Glomus and Ambispora present in Sequoiadendron roots. Overall, we found that AM fungal colonization of giant sequoia roots was associated with availability of plant-assimilated carbon to the fungus rather than with the AM fungal supply of mineral nutrients to the roots. We conclude that restoration practices affecting light availability and carbon assimilation alter feedbacks between sapling growth and activity of AM fungi in the roots.