Bacterial communities associated with the lichen symbiosis

Lichens are commonly described as a mutualistic symbiosis between fungi and "algae" (Chlorophyta or Cyanobacteria); however, they also have internal bacterial communities. Recent research suggests that lichen-associated microbes are an integral component of lichen thalli and that the classical view of this symbiotic relationship should be expanded to include bacteria. However, we still have a limited understanding of the phylogenetic structure of these communities and their variability across lichen species. To address these knowledge gaps, we used bar-coded pyrosequencing to survey the bacterial communities associated with lichens. Bacterial sequences obtained from four lichen species at multiple locations on rock outcrops suggested that each lichen species harbored a distinct community and that all communities were dominated by Alphaproteobacteria. Across all samples, we recovered numerous bacterial phylotypes that were closely related to sequences isolated from lichens in prior investigations, including those from a lichen-associated Rhizobiales lineage (LAR1; putative N(2) fixers). LAR1-related phylotypes were relatively abundant and were found in all four lichen species, and many sequences closely related to other known N(2) fixers (e.g., Azospirillum, Bradyrhizobium, and Frankia) were recovered. Our findings confirm the presence of highly structured bacterial communities within lichens and provide additional evidence that these bacteria may serve distinct functional roles within lichen symbioses.     

Compatibility and thigmotropism in the lichen symbiosis: A reappraisal

The development of many complex stratified lichen thalli is made through stages of complex phenotypic interactions between a filamentous fungus (the mycobiont), and a trebouxioid alga (the photobiont). Typically, the second stage of this symbiotic development is marked by the envelopment of the photobiont by the mycobiont through increased lateral hyphal branching and the formation of appressoria. Previously, the mycobiont’s envelopment of photobiont cells was considered thigmotropic (a growth response due to shape) as a mycobiont can envelop algal sized objects in its environment. However, after growing the mycobiontCladonia grayi with various phototrophs and glass beads, we conclude that the mycobiont does not show this characteristic second stage morphological response when grown in non-compatible pairings. Instead,C. grayi displays a distinctive morphological growth response only in compatible symbiotic pairings, such as with its natural photobiontAsterochlor’is sp.     

Impact of temperature on the arbuscular mycorrhizal (AM) symbiosis: growth responses of the host plant and its AM fungal partner

The growth response of the hyphae of mycorrhizal fungi has been determined, both when plant and fungus together and when only the fungus was exposed to a temperature change. Two host plant species, Plantago lanceolata and Holcus lanatus, were grown separately in pots inoculated with the mycorrhizal fungus Glomus mosseae at 20/18 degrees C (day/night); half of the pots were then transferred to 12/10 degrees C. Plant and fungal growth were determined at six sequential destructive harvests. A second experiment investigated the direct effect of temperature on the length of the extra-radical mycelium (ERM) of three mycorrhizal fungal species. Growth boxes were divided in two equal compartments by a 20 micro m mesh, allowing only the ERM and not roots to grow into a fungal compartment, which was either heated (+8 degrees C) or kept at ambient temperature. ERM length (LERM) was determined on five sampling dates. Growth of H. lanatus was little affected by temperature, whereas growth of P. lanceolata increased with temperature, and both specific leaf area (SLA) and specific root length (SRL) increased independently of plant size. Percentage of colonized root (LRC) and LERM were positively correlated with temperature when in symbiosis with P. lanceolata, but differences in LRC were a function of plant biomass. Colonization was very low in H. lanatus roots and there was no significant temperature effect. In the fungal compartment LERM increased over time and was greatest for Glomus mosseae. Heating the fungal compartment significantly increased LERM in two of the three species but did not affect LRC. However, it significantly increased SRL of roots in the plant compartment, suggesting that the fungus plays a regulatory role in the growth dynamics of the symbiosis. These temperature responses have implications for modelling carbon dynamics under global climate change.     

Twenty-seven modes of reproduction in the obligate lichen symbiosis

Fungi exhibit some of the greatest reproductive diversity across Eukaryotes. In addition to sexual and asexual reproduction, fungi engage in parasexual (mitotic recombinatorial) processes to acquire new genetic variation. Reproduction has been studied extensively in numerous free-living fungi but comparatively less knowledge exists for lichenized fungi, which are assumed to reproduce only through sexual spores, asexual conidia, and specialized asexual lichen propagules. We present a new conceptual framework describing reproductive modes in lichens that includes sexual and asexual processes as well as accommodating the possibility of parasexual reproduction. To support the plausibility of some of these modes of reproduction, we reviewed data spanning more than 200 years of anatomical investigation. We recovered evidence supporting the possibility of 22 of 27 possible modes of reproduction and found no counter-evidence to suggest the remaining five do not occur in nature. This conceptual framework allows for a greater plurality of reproductive processes than previously acknowledged in lichens, exceeding that of their non-lichenized relatives.     

Temperature transcends partner specificity in the symbiosis establishment of a cnidarian

Coral reef research has predominantly focused on the effect of temperature on the breakdown of coral-dinoflagellate symbioses. However, less is known about how increasing temperature affects the establishment of new coral-dinoflagellate associations. Inter-partner specificity and environment-dependent colonization are two constraints proposed to limit the acquisition of more heat tolerant symbionts. Here, we investigated the symbiotic dynamics of various photosymbionts in different host genotypes under “optimal” and elevated temperature conditions. To do this, we inoculated symbiont-free polyps of the sea anemone Exaiptasia pallida originating from Hawaii (H2), North Carolina (CC7), and the Red Sea (RS) with the same mixture of native symbiont strains (Breviolum minutum, Symbiodinium linucheae, S. microadriaticum, and a Breviolum type from the Red Sea) at 25 and 32 °C, and assessed their ITS2 composition, colonization rates, and PSII photochemical efficiency (Fv/Fm). Symbiont communities across thermal conditions differed significantly for all hosts, suggesting that temperature rather than partner specificity had a stronger effect on symbiosis establishment. Overall, we detected higher abundances of more heat resistant Symbiodiniaceae types in the 32 °C treatments. Our data further showed that PSII photophysiology under elevated temperature improved with thermal pre-exposure (i.e., higher Fv/Fm), yet, this effect depended on host genotype and was influenced by active feeding as photochemical efficiency dropped in response to food deprivation. These findings highlight the role of temperature and partner fidelity in the establishment and performance of symbiosis and demonstrate the importance of heterotrophy for symbiotic cnidarians to endure and recover from stress.Subject terms: Climate-change ecology, Animal physiology     

Nitric oxide is required for an optimal establishment of the Medicago truncatula-Sinorhizobium meliloti symbiosis

Nitric oxide (NO) is a gaseous molecule that participates in numerous plant signalling pathways. It is involved in plant responses to pathogens and development processes such as seed germination, flowering and stomatal closure. Using a permeable NO-specific fluorescent probe and a bacterial reporter strain expressing the lacZ gene under the control of a NO-responsive promoter, we detected NO production in the first steps, during infection threads growth, of the Medicago truncatula-Sinorhizobium meliloti symbiotic interaction. Nitric oxide was also detected, by confocal microscopy, in nodule primordia. Depletion of NO caused by cPTIO (2-(4-carboxyphenyl)-4,4,5,5-tetramethyl imidazoline-1-oxyl-3-oxide), an NO scavenger, resulted in a significant delay in nodule appearance. The overexpression of a bacterial hmp gene, encoding a flavohaemoglobin able to scavenge NO, under the control of a nodule-specific promoter (pENOD20) in transgenic roots, led to the same phenotype. The NO scavenging resulting from these approaches provoked the downregulation of plant genes involved in nodule development, such as MtCRE1 and MtCCS52A. Furthermore, an Hmp-overexpressing S. meliloti mutant strain was found to be less competitive than the wild type in the nodulation process. Taken together, these results indicate that NO is required for an optimal establishment of the M. truncatula-S. meliloti symbiotic interaction.     

Evolution of legume-rhizobium symbiosis for an improved ecological efficiency and genotypic specificity of partner interactions

Mathematical simulation of the evolution of polymorphic legume-rhizobium symbiosis showed that co-evolution of the partners for an improved ecological efficiency of symbiosis is greatly stimulated when low-active N₂-fixing and non-N₂-fixing strains of nodule bacteria are prohibited from colonizing nodules. The results of analysis of the model were collated with the comparative morphology of the infection process in various legumes, and its was assumed that mechanisms controlling bacterial reproduction in nodules arose in early evolution of symbiosis in primitive legumes owing to a transition from mixed to clonal infection. The development of such mechanisms was associated with adaptively valuable macroevolutionary transformations of symbiosis and directed its microevolution towards a parallel increase in the specificity and efficiency of mutualism. The increase was due to a reorganization of selective processes in endosymbiotic bacterial populations, which was based on changes in their genetic and spatial structures and optimized metabolic feedbacks between the partners (preferential allocation of photosynthesis products to the most active N₂-fixing strains).     

Bioactive lichen metabolites: alpine habitats as an untapped source

Lichens are fungal and algal/cyanobacterial symbioses resulting in the production of specific metabolites. Some of these are forming an available biomass for phytochemical investigations, including the assessment of biological activities of the isolated compounds. The alpine or polar region are characterised by highly stressful environmental conditions for many organisms, but lichens are among the dominating organisms in these habitats. In the performant mutual protective system, lichen fungi often accumulate high amounts of metabolites with specific physicochemical properties (UV absorbents, hydrophobicity) which help the lichens to survive. Unique secondary metabolites and polysaccharides have been isolated and tested from these organisms. Even though this has been tested until now only with a low number of compounds so far, interesting activities have been recorded. We review here some of the antimicrobial, anti-inflammatory, antiproliferative and antioxidant activities properties described. Solutions with axenic biotechnological cultivation of each symbiotic partner and particularly the mycobiont to obtain the lichen secondary metabolites are challenging to overcome the limitations for the supply of these rare compounds. Additionally, these lichens appear to harbour a diversity of culturable microorganisms from which active compounds have also been isolated recently.     

Microbial consortia of bacteria and fungi with focus on the lichen symbiosis

The investigation of fungal–bacterial interactions is an emerging field of research applying tools of modern microbial ecology. Studies have previously focused on the mycorrhizosphere, but in past decade, the role of bacteria in other fungal niches has been increasingly evaluated. This review presents recent progress in the understanding of fungal–bacterial interactions and contains a special focus on lichen symbioses. Lichens are traditionally considered as mutualisms between fungi and photoautotrophic species, but recent molecular approaches have revealed that lichens also harbour diverse microbial communities. Using modern DNA/RNA-based and microscopic techniques (e.g. FISH and confocal laser scanning microscopy) we are now able to analyse the abundance, composition, and structure of microbial communities in the lichen holobiont. Lichen-associated microbial communities consist of diverse taxonomic groups; the majority of bacteria belong to Alphaproteobacteria. Microbial communities can form biofilm-like structures on specific parts of the lichen thallus. Until now, the function and interaction within the microbial consortia is not fully understood. The functions displayed mainly by culturable strains suggest that bacteria have lytic activities, complement the nitrogen budget and produce bioactive substances, including hormones and antibiotics. Bacterial contribution to the lichen symbiosis is perhaps not restricted to one particular function in the lichen system, but supports a complex functional network which remains to be studied in greater detail.     

Hemostasis as an optimal system

Zymogen and procofactor concentrations in physiological biochemical systems (PBS) have not yet been explained. The problem in question is to determine optimal plasma clotting factor (factors II, VII, IX, and X and cofactors V and VIII) concentrations for coagulation system (CS) as a whole. Constrained optimization technique is used to solve this problem. The constraint is determined by the ability of the CS to perform its physiological function--thrombin generation (and hence clot formation)--under vessel injury conditions. The constraint statement is based on the CS dynamics equations. In solving the problem the Lagrange multiplier is used. A hypothesis is advanced that this problem can be solved based on principle of minimum protein consumption subject to an above constraint. The results obtained indicate that the optimal clotting factor concentrations are in good agreement with those measured by biochemical techniques. A comparison between the theoretical results and experimental data lends support for our hypothesis that zymogen and procofactor concentrations in the CS (and, probably, for other biochemical systems) are determined by the principle of minimum protein consumption.     

Viscosity as an inseparable partner of muscle contraction

A simple model is presented where, by an iterative procedure, the forces delivered by the power strokes are summed up to overcome the load. The system is moderated by the viscous hindrance. The model reproduces the features of muscle contraction as defined by the data of He et al. [1997. ATPase kinetics on activation of permeabilized isometric fibres from rabbit and frog muscle: a real time phosphate assay. J. Physiol. 501, 125-148] on rabbit psoas muscle fibres. According to the model power strokes are random. Energy summation take place if the subsequent power stroke occurs before the energy delivered by the previous power stroke is completely used. In order the sarcomere to be competent to contract initial driving force must reach a threshold whose value increases with the load. The step size of the power stroke decreases with the increase of the load. The viscous regime is simulated by the equation, where 1/k measures the viscous hindrance of the system. The relationship between water activity, viscosity and stiffness is discussed. It is concluded that the three parameters vary cyclically and that when water activity decreases (sarcomere shortening, cross-bridge attachment) viscosity and stiffness increase.     

First report on polysaccharides of Asterochloris and their potential role in the lichen symbiosis

A structural study of the carbohydrates from the aposymbiotically cultured Asterochloris sp., the algal symbiont of the lichen Cladina confusa was carried out for the first time. A xylorhamnogalactofuranan was purified and was predominated by (1-->3)-linked galactofuranosyl units with sidechains in position 6 on approximately 6.4% of the units. The sidechains have galactofuranosyl units 5-O and 6-O-substituted, as well rhamnopyranosyl units 2-O, 3-O and 2,3-di-O-substituted. Xylose was detected only as nonreducing end units, together with galactofuranosyl units. Amylose and a beta-(1-->4)-xylan were also present. These polysaccharides have not been found in the symbiotic thallus of C. confusa, which contained only glucans, galactomannoglucan and galactoglucomannan. A potential role of these carbohydrates in lichen recognition proccess is also discussed.     

Recognition events in AM symbiosis: analysis of fungal gene expression at the early appressorium stage

Arbuscular mycorrhizal symbiosis is induced upon a series of recognition events involving the reorganization of both plant and fungal cellular programs culminating in the formation of appressoria on the epidermal root cells. In this work we monitored for the first time the genetic changes occurring in the fungal partner during early appressorium development. We established an in vitro system of Glomus mosseae and Petroselinum crispum for studying appressorium formation and found that after 120 h first appressoria developed in the root epidermis. We have constructed a fungal subtractive suppressive library enriched in genes up-regulated at this stage. Our aim was to identify early signaling events during plant recognition leading to appressoria formation. The library contains 375 clones with an average size of 500 bp. From these, 200 clones were sequenced and most of them represent gene fragments with no known homologues (63%) and therefore putative new genes specific to the mycorrhiza symbiosis. Reverse-Northern blot and RT-PCR analyses confirmed that ca. 30% of the genes present in the library were up-regulated upon plant induction after 120 h. Among the genes with homologues in other organisms we found several genes common to other plant-microbe interactions including some genes related to Ca2+-dependent signaling. The up-regulation of these genes opens the possibility that Ca2+ plays a role in the early stages of mycorrhiza formation as it has been found in other plant-microbe interactions such as the Rhizobium symbiosis or the Magnaporthe grisea/rice pathogenic interaction.     

Dating in the dark: how roots respond to fungal signals to establish arbuscular mycorrhizal symbiosis

The arbuscular mycorrhizal symbiosis that involves most plants and Glomeromycota fungi is the result of a complex exchange of molecular information, which commences before the partners are in physical contact. On the one hand, plants release soluble factors, including strigolactones that activate both the metabolism and branching of the fungal partners. On the other hand, fungi use compounds that trigger the signaling transduction pathways that are required for the symbiotic modus of plant cells. Here we describe some of the recent discoveries regarding the fungal molecules involved in rhizospheric conversation, and the way in which they are perceived by their hosts. We conclude that similar signaling molecules may have different meanings, depending on the context. However, at the end, specificity must be maintained to ensure appropriate partners enter symbiosis.