Divergent colonization traits, convergent benefits: different species of arbuscular mycorrhizal fungi alleviate Meloidogyne incognita damage in tomato.

Arbuscular mycorrhizal fungi (AMF) can increase plant tolerance and/or resistance to pests such as the root-knot nematode Meloidogyne incognita. However, the ameliorative effects may depend on AMF species. The aim of this work was therefore to evaluate whether four AMF species differentially affect plant performance in response to M. incognita infection. Tomato plants grown in greenhouse conditions were inoculated with four different AMF isolates (Claroideoglomus claroideum, Funneliformis mosseae, Gigaspora margarita, and Rhizophagus intraradices) and infected with 100 second stage juveniles of M. incognita at two different times: simultaneously or 2 weeks after the inoculation with AMF. After 60 days, the number of galls, egg masses, and reproduction factor of the nematodes were assessed along with plant biomass, phosphorus (P), and nitrogen concentrations in roots and shoots and root colonization by AMF. Only the simultaneous nematode inoculation without AMF caused a large reduction in plant shoot biomass, while all AMF species were able to ameliorate this effect and improve plant P uptake. The AMF isolates responded differently to the interaction with nematodes, either increasing the frequency of vesicles (C. claroideum) or reducing the number of arbuscules (F. mosseae and Gi. margarita). AMF inoculation did not decrease galls; however, it reduced the number of egg masses per gall in nematode simultaneous inoculation, except for C. claroideum. This work shows the importance of biotic stress alleviation associated with an improvement in P uptake and mediated by four different AMF species, irrespective of their fungal root colonization levels and specific interactions with the parasite.

Non-typeable Haemophilus influenzae major outer membrane protein P5 contributes to bacterial membrane stability, and affects the membrane protein composition crucial for interactions with the human host.

Non-typeable Haemophilus influenzae (NTHi) is a Gram-negative human pathogen that causes a wide range of airway diseases. NTHi has a plethora of mechanisms to colonize while evading the host immune system for the establishment of infection. We previously showed that the outer membrane protein P5 contributes to bacterial serum resistance by the recruitment of complement regulators. Here, we report a novel role of P5 in maintaining bacterial outer membrane (OM) integrity and protein composition important for NTHi-host interactions. In silico analysis revealed a peptidoglycan-binding motif at the periplasmic C-terminal domain (CTD) of P5. In a peptidoglycan-binding assay, the CTD of P5 (P5CTD) formed a complex with peptidoglycan. Protein profiling analysis revealed that deletion of CTD or the entire P5 changed the membrane protein composition of the strains NTHi 3655Δp5CTD and NTHi 3655Δp5, respectively. Relative abundance of several membrane-associated virulence factors that are crucial for adherence to the airway mucosa, and serum resistance were altered. This was also supported by similar attenuated pathogenic phenotypes observed in both NTHi 3655Δp5 CTD and NTHi 3655Δp5. We found (i) a decreased adherence to airway epithelial cells and fibronectin, (ii) increased complement-mediated killing, and (iii) increased sensitivity to the ß-lactam antibiotics in both mutants compared to NTHi 3655 wild-type. These mutants were also more sensitive to lysis at hyperosmotic conditions and hypervesiculated compared to the parent wild-type bacteria. In conclusion, our results suggest that P5 is important for bacterial OM stability, which ultimately affects the membrane proteome and NTHi pathogenesis.

Protein domain-dependent vesiculation of Lipoprotein A, a protein that is important in cell wall synthesis and fitness of the human respiratory pathogen Haemophilus influenzae.

The human pathogen Haemophilus influenzae causes respiratory tract infections and is commonly associated with prolonged carriage in patients with chronic obstructive pulmonary disease. Production of outer membrane vesicles (OMVs) is a ubiquitous phenomenon observed in Gram-negative bacteria including H. influenzae. OMVs play an important role in various interactions with the human host; from neutralization of antibodies and complement activation to spread of antimicrobial resistance. Upon vesiculation certain proteins are found in OMVs and some proteins are retained at the cell membrane. The mechanism for this phenomenon is not fully elucidated. We employed mass spectrometry to study vesiculation and the fate of proteins in the outer membrane. Functional groups of proteins were differentially distributed on the cell surface and in OMVs. Despite its supposedly periplasmic and outer membrane location, we found that the peptidoglycan synthase-activator Lipoprotein A (LpoA) was accumulated in OMVs relative to membrane fractions. A mutant devoid of LpoA lost its fitness as revealed by growth and electron microscopy. Furthermore, high-pressure liquid chromatography disclosed a lower concentration (55%) of peptidoglycan in the LpoA-deficient H. influenzae compared to the parent wild type bacterium. Using an LpoA-mNeonGreen fusion protein and fluorescence microscopy, we observed that LpoA was enriched in "foci" in the cell envelope, and further located in the septum during cell division. To define the fate of LpoA, C-terminally truncated LpoA-variants were constructed, and we found that the LpoA C-terminal domain promoted optimal transportation to the OMVs as revealed by flow cytometry. Taken together, our study highlights the importance of LpoA for H. influenzae peptidoglycan biogenesis and provides novel insights into cell wall integrity and OMV production.

Asymmetric Interaction Between Two Mycorrhizal Fungal Guilds and Consequences for the Establishment of Their Host Plants.

Arbuscular mycorrhiza (AM) and ectomycorrhiza (EcM) are the most abundant and widespread types of mycorrhizal symbiosis, but there is little and sometimes conflicting information regarding the interaction between AM fungi (AMF) and EcM fungi (EcMF) in soils. Their competition for resources can be particularly relevant in successional ecosystems, which usually present a transition from AM-forming herbaceous vegetation to EcM-forming woody species. The aims of this study were to describe the interaction between mycorrhizal fungal communities associated with AM and EcM hosts naturally coexisting during primary succession on spoil banks and to evaluate how this interaction affects growth and mycorrhizal colonization of seedlings of both species. We conducted a greenhouse microcosm experiment with Betula pendula and Hieracium caespitosum as EcM and AM hosts, respectively. They were cultivated in three-compartment rhizoboxes. Two lateral compartments contained different combinations of both host plants as sources of fungal mycelia colonizing the middle compartment, where fungal biomass, diversity, and community composition as well as the growth of each host plant species' seedlings were analyzed. The study's main finding was an asymmetric outcome of the interaction between the two plant species: while H. caespitosum and associated AMF reduced the abundance of EcMF in soil, modified the composition of EcMF communities, and also tended to decrease growth and mycorrhizal colonization of B. pendula seedlings, the EcM host did not have such effects on AM plants and associated AMF. In the context of primary succession, these findings suggest that ruderal AM hosts could hinder the development of EcM tree seedlings, thus slowing the transition from AM-dominated to EcM-dominated vegetation in early successional stages.

Ms1 RNA Interacts With the RNA Polymerase Core in Streptomyces coelicolor and Was Identified in Majority of Actinobacteria Using a Linguistic Gene Synteny Search.

Bacteria employ small non-coding RNAs (sRNAs) to regulate gene expression. Ms1 is an sRNA that binds to the RNA polymerase (RNAP) core and affects the intracellular level of this essential enzyme. Ms1 is structurally related to 6S RNA that binds to a different form of RNAP, the holoenzyme bearing the primary sigma factor. 6S RNAs are widespread in the bacterial kingdom except for the industrially and medicinally important Actinobacteria. While Ms1 RNA was identified in Mycobacterium, it is not clear whether Ms1 RNA is present also in other Actinobacteria species. Here, using a computational search based on secondary structure similarities combined with a linguistic gene synteny approach, we identified Ms1 RNA in Streptomyces. In S. coelicolor, Ms1 RNA overlaps with the previously annotated scr3559 sRNA with an unknown function. We experimentally confirmed that Ms1 RNA/scr3559 associates with the RNAP core without the primary sigma factor HrdB in vivo. Subsequently, we applied the computational approach to other Actinobacteria and identified Ms1 RNA candidates in 824 Actinobacteria species, revealing Ms1 RNA as a widespread class of RNAP binding sRNAs, and demonstrating the ability of our multifactorial computational approach to identify weakly conserved sRNAs in evolutionarily distant genomes.

Gene Expression Regulation in Airway Pathogens: Importance for Otitis Media.

Otitis media (OM) is an inflammatory disorder in the middle ear. It is mainly caused by viruses or bacteria associated with the airways. Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis are the three main pathogens in infection-related OM, especially in younger children. In this review, we will focus upon the multifaceted gene regulation mechanisms that are well-orchestrated in S. pneumoniae, H. influenzae, and M. catarrhalis during the course of infection in the middle ear either in experimental OM or in clinical settings. The sophisticated findings from the past 10 years on how the othopathogens govern their virulence phenotypes for survival and host adaptation via phase variation- and quorum sensing-dependent gene regulation, will be systematically discussed. Comprehensive understanding of gene expression regulation mechanisms employed by pathogens during the onset of OM may provide new insights for the design of a new generation of antimicrobial agents in the fight against bacterial pathogens while combating the serious emergence of antimicrobial resistance.

The effects of arbuscular mycorrhizal fungal species and taxonomic groups on stressed and unstressed plants: a global meta-analysis.

The great majority of plants gain access to soil nutrients and enhance their performance under stressful conditions through symbiosis with arbuscular mycorrhizal fungi (AMF). The benefits that AMF confer vary among species and taxonomic groups. However, a comparative analysis of the different benefits among AMF has not yet been performed. We conducted a global meta-analysis of recent studies testing the benefits of individual AMF species and main taxonomic groups in terms of plant performance (growth and nutrition). Separately, we examined AMF benefits to plants facing biotic (pathogens, parasites, and herbivores) and abiotic (drought, salinity, and heavy metals) stress. AMF had stronger positive effects on phosphorus nutrition than on plant growth and nitrogen nutrition and the effects on the growth of plants facing biotic and abiotic stresses were similarly positive. While the AMF taxonomic groups showed positive effects on plant performance either with or without stress, Diversisporales were the most beneficial to plants without stress and Gigasporales to plants facing biotic stress. Our results provide a comprehensive analysis of the benefits of different AMF species and taxonomic groups on plant performance and useful insights for their management and use as bio-inoculants for agriculture and restoration.

Strigolactones: New players in the nitrogen-phosphorus signalling interplay.

Nitrogen (N) and phosphorus (P) are among the most important macronutrients for plant growth and development, and the most widely used as fertilizers. Understanding how plants sense and respond to N and P deficiency is essential to optimize and reduce the use of chemical fertilizers. Strigolactones (SLs) are phytohormones acting as modulators and sensors of plant responses to P deficiency. In the present work, we assess the potential role of SLs in N starvation and in the N-P signalling interplay. Physiological, transcriptional and metabolic responses were analysed in wild-type and SL-deficient tomato plants grown under different P and N regimes, and in plants treated with a short-term pulse of the synthetic SL analogue 2'-epi-GR24. The results evidence that plants prioritize N over P status by affecting SL biosynthesis. We also show that SLs modulate the expression of key regulatory genes of phosphate and nitrate signalling pathways, including the N-P integrators PHO2 and NIGT1/HHO. The results support a key role for SLs as sensors during early plant responses to both N and phosphate starvation and mediating the N-P signalling interplay, indicating that SLs are involved in more physiological processes than so far proposed.

Mycobacterial HelD is a nucleic acids-clearing factor for RNA polymerase.

RNA synthesis is central to life, and RNA polymerase (RNAP) depends on accessory factors for recovery from stalled states and adaptation to environmental changes. Here, we investigated the mechanism by which a helicase-like factor HelD recycles RNAP. We report a cryo-EM structure of a complex between the Mycobacterium smegmatis RNAP and HelD. The crescent-shaped HelD simultaneously penetrates deep into two RNAP channels that are responsible for nucleic acids binding and substrate delivery to the active site, thereby locking RNAP in an inactive state. We show that HelD prevents non-specific interactions between RNAP and DNA and dissociates stalled transcription elongation complexes. The liberated RNAP can either stay dormant, sequestered by HelD, or upon HelD release, restart transcription. Our results provide insights into the architecture and regulation of the highly medically-relevant mycobacterial transcription machinery and define HelD as a clearing factor that releases RNAP from nonfunctional complexes with nucleic acids.

Molecular Quantification of Arbuscular Mycorrhizal Fungal Root Colonization.

Specific quantification of root-colonizing arbuscular mycorrhizal fungi (AMF) by quantitative real-time PCR is a high-throughput technique, most suitable for determining abundances of AMF species or isolates in previously characterized experimental systems. The principal steps are the choice and validation of an appropriate assay to specifically amplify a gene fragment of the target AMF, preparation of templates from root samples, and quantification of the fungal gene copy numbers in these templates. The use of a suitable assay is crucial for a correct data collection but also highly specific for each experimental system and is therefore covered by general recommendations. Subsequently, specific steps are described for the validation of the assay using a standard dilution series, the determination of appropriate dilutions of DNA extracts from roots, and the quantification of the gene copy numbers in samples including calculations.

Switching transcription with bacterial RNA polymerase through photocaging, photorelease and phosphorylation reactions in the major groove of DNA.

We report proof of principle biomimetic switching of transcription in vitro through non-natural chemical reactions in the major groove of DNA templates. Photocaged DNA templates containing nitrobenzyl-protected 5-hydroxymethyluracil or - cytosine permitted no transcription with E. coli RNA polymerase (OFF state). Their irradiation with 400 nm light resulted in DNA templates containing hydroxymethylpyrimidines, which switched transcription ON with a higher yield (250-350%) compared to non-modified DNA. Phosphorylation of templates containing 5-hydroxymethyluracil (but not 5-hydroxymethylcytosine) then turned transcription OFF again. It is the first step towards artificial bioorthogonal chemical epigenetics.

Abiotic contexts consistently influence mycorrhiza functioning independently of the composition of synthetic arbuscular mycorrhizal fungal communities.

The relationship between mycorrhiza functioning and composition of arbuscular mycorrhizal (AM) fungal communities is an important but experimentally still rather little explored topic. The main aim of this study was thus to link magnitude of plant benefits from AM symbiosis in different abiotic contexts with quantitative changes in AM fungal community composition. A synthetic AM fungal community inoculated to the model host plant Medicago truncatula was exposed to four different abiotic contexts, namely drought, elevated phosphorus availability, and shading, as compared to standard cultivation conditions, for two cultivation cycles. Growth and phosphorus uptake of the host plants was evaluated along with the quantitative composition of the synthetic AM fungal community. Abiotic context consistently influenced mycorrhiza functioning in terms of plant benefits, and the effects were clearly linked to the P requirement of non-inoculated control plants. In contrast, the abiotic context only had a small and transient effect on the quantitative AM fungal community composition. Our findings suggest no relationship between the degree of mutualism in AM symbiosis and the relative abundances of AM fungal species in communities in our simplified model system. The observed progressive dominance of one AM fungal species indicates an important role of different growth rates of AM fungal species for the establishment of AM fungal communities in simplified systems such as agroecosystems.

Ms1 RNA increases the amount of RNA polymerase in Mycobacterium smegmatis.

Ms1 is a sRNA recently found in mycobacteria and several other actinobacterial species. Ms1 interacts with the RNA polymerase (RNAP) core devoid of sigma factors, which differs from 6S RNA that binds to RNAP holoenzymes containing the primary sigma factor. Here we show that Ms1 is the most abundant non-rRNA transcript in stationary phase in Mycobacterium smegmatis. The accumulation of Ms1 stems from its high-level synthesis combined with decreased degradation. We identify the Ms1 promoter, PMs1 , and cis-acting elements important for its activity. Furthermore, we demonstrate that PNPase (an RNase) contributes to the differential accumulation of Ms1 during growth. Then, by comparing the transcriptomes of wt and ΔMs1 strains from stationary phase, we reveal that Ms1 affects the intracellular level of RNAP. The absence of Ms1 results in decreased levels of the mRNAs encoding ß and ß' subunits of RNAP, which is also reflected at the protein level. Thus, the ΔMs1 strain has a smaller pool of RNAPs available when the transcriptional demand increases. This contributes to the inability of the ΔMs1 strain to rapidly react to environmental changes during outgrowth from stationary phase.

Turning Off Transcription with Bacterial RNA Polymerase through CuAAC Click Reactions of DNA Containing 5-Ethynyluracil.

Copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) click reaction in the major groove of DNA containing 5-ethynyluracil (UE ) with azides was used for turning off sequence-specific protein-DNA interactions. The concept was first demonstrated on switching off cleavage of short modified DNA by restriction endonuclease BamHI-HF. Finally, DNA template containing UE was used for in vitro transcription with E. coli RNA polymerase and the transcription was turned off by CuAAC with 3-azidopropane-1,2-diol or 3-azido-7-hydroxycoumarin.

Arbuscular mycorrhizal fungi and associated microbial communities from dry grassland do not improve plant growth on abandoned field soil.

After abandonment of agricultural fields, some grassland plant species colonize these sites with a frequency equivalent to dry grasslands (generalists) while others are missing or underrepresented in abandoned fields (specialists). We aimed to understand the inability of specialists to spread on abandoned fields by exploring whether performance of generalists and specialists depended on soil abiotic and/or biotic legacy. We performed a greenhouse experiment with 12 species, six specialists and six generalists. The plants were grown in sterile soil from dry grassland or abandoned field inoculated with microbial communities from one or the other site. Plant growth, abundance of mycorrhizal structures and plant response to inoculation were evaluated. We focused on arbuscular mycorrhizal fungi (AMF), one of the most important parts of soil communities affecting plant performance. The abandoned field soil negatively affected plant growth, but positively affected plant response to inoculation. The AMF community from both sites differed in infectivity and taxa frequencies. The lower AMF taxa frequency in the dry grassland soil suggested a lack of functional complementarity. Despite the fact that dry grassland AMF produced more arbuscules, the dry grassland inoculum did not improve phosphorus nutrition of specialists contrary to the abandoned field inoculum. Inoculum origin did not affect phosphorus nutrition of generalists. The lower effectiveness of the dry grassland microbial community toward plant performance excludes its inoculation in the abandoned field soil as a solution to allow settlement of specialists. Still, the distinct response of specialists and generalists to inoculation suggested that they differ in AMF responsiveness.

5-(Hydroxymethyl)uracil and -cytosine as potential epigenetic marks enhancing or inhibiting transcription with bacterial RNA polymerase.

DNA templates containing 5-hydroxymethyluracil or 5-hydroxymethylcytosine were used in an in vitro transcription assay with RNA polymerase from Escherichia coli. A strong enhancement of transcription was observed from DNA containing the Pveg promoter whereas a decrease was observed from DNA containing the rrnB P1 promoter, suggesting that they may act as epigenetic marks.

Inoculation effects on root-colonizing arbuscular mycorrhizal fungal communities spread beyond directly inoculated plants.

Inoculation with arbuscular mycorrhizal fungi (AMF) may improve plant performance at disturbed sites, but inoculation may also suppress root colonization by native AMF and decrease the diversity of the root-colonizing AMF community. This has been shown for the roots of directly inoculated plants, but little is known about the stability of inoculation effects, and to which degree the inoculant and the inoculation-induced changes in AMF community composition spread into newly emerging seedlings that were not in direct contact with the introduced propagules. We addressed this topic in a greenhouse experiment based on the soil and native AMF community of a post-mining site. Plants were cultivated in compartmented pots with substrate containing the native AMF community, where AMF extraradical mycelium radiating from directly inoculated plants was allowed to inoculate neighboring plants. The abundances of the inoculated isolate and of native AMF taxa were monitored in the roots of the directly inoculated plants and the neighboring plants by quantitative real-time PCR. As expected, inoculation suppressed root colonization of the directly inoculated plants by other AMF taxa of the native AMF community and also by native genotypes of the same species as used for inoculation. In the neighboring plants, high abundance of the inoculant and the suppression of native AMF were maintained. Thus, we demonstrate that inoculation effects on native AMF propagate into plants that were not in direct contact with the introduced inoculum, and are therefore likely to persist at the site of inoculation.

Real-time PCR quantification of arbuscular mycorrhizal fungi: does the use of nuclear or mitochondrial markers make a difference?

Root colonization by arbuscular mycorrhizal fungi (AMF) can be quantified by different approaches. We compared two approaches that enable discrimination of specific AMF taxa and are therefore emerging as alternative to most commonly performed microscopic quantification of AMF in roots: quantitative real-time PCR (qPCR) using markers in nuclear ribosomal DNA (nrDNA) and mitochondrial ribosomal DNA (mtDNA). In a greenhouse experiment, Medicago truncatula was inoculated with four isolates belonging to different AMF species (Rhizophagus irregularis, Claroideoglomus claroideum, Gigaspora margarita and Funneliformis mosseae). The AMF were quantified in the root samples by qPCR targeted to both markers, microscopy and contents of AMF-specific phospholipid fatty acids (PLFA). Copy numbers of nrDNA and mtDNA were closely related within all isolates; however, the slopes and intercepts of the linear relationships significantly differed among the isolates. Across all isolates, a large proportion of variance in nrDNA copy numbers was explained by root colonization intensity or contents of AMF-specific PLFA, while variance in mtDNA copy numbers was mainly explained by differences among AMF isolates. We propose that the encountered inter-isolate differences in the ratios of mtDNA and nrDNA copy numbers reflect different physiological states of the isolates. Our results suggest that nrDNA is a more suitable marker region than mtDNA for the quantification of multiple AMF taxa as its copy numbers are better related to fungal biomass across taxa than are copy numbers of mtDNA.

Plant Communities Rather than Soil Properties Structure Arbuscular Mycorrhizal Fungal Communities along Primary Succession on a Mine Spoil.

Arbuscular mycorrhizal fungal (AMF) community assembly during primary succession has so far received little attention. It remains therefore unclear, which of the factors, driving AMF community composition, are important during ecosystem development. We addressed this question on a large spoil heap, which provides a mosaic of sites in different successional stages under different managements. We selected 24 sites of c. 12, 20, 30, or 50 years in age, including sites with spontaneously developing vegetation and sites reclaimed by alder plantations. On each site, we sampled twice a year roots of the perennial rhizomatous grass Calamagrostis epigejos (Poaceae) to determine AMF root colonization and diversity (using 454-sequencing), determined the soil chemical properties and composition of plant communities. AMF taxa richness was unaffected by site age, but AMF composition variation increased along the chronosequences. AMF communities were unaffected by soil chemistry, but related to the composition of neighboring plant communities of the sampled C. epigejos plants. In contrast, the plant communities of the sites were more distinctively structured than the AMF communities along the four successional stages. We conclude that AMF and plant community successions respond to different factors. AMF communities seem to be influenced by biotic rather than by abiotic factors and to diverge with successional age.

Arbuscular Mycorrhiza Stimulates Biological Nitrogen Fixation in Two Medicago spp. through Improved Phosphorus Acquisition.

Legumes establish root symbioses with rhizobia that provide plants with nitrogen (N) through biological N fixation (BNF), as well as with arbuscular mycorrhizal (AM) fungi that mediate improved plant phosphorus (P) uptake. Such complex relationships complicate our understanding of nutrient acquisition by legumes and how they reward their symbiotic partners with carbon along gradients of environmental conditions. In order to disentangle the interplay between BNF and AM symbioses in two Medicago species (Medicago truncatula and M. sativa) along a P-fertilization gradient, we conducted a pot experiment where the rhizobia-treated plants were either inoculated or not inoculated with AM fungus Rhizophagus irregularis 'PH5' and grown in two nutrient-poor substrates subjected to one of three different P-supply levels. Throughout the experiment, all plants were fertilized with 15N-enriched liquid N-fertilizer to allow for assessment of BNF efficiency in terms of the fraction of N in the plants derived from the BNF (%NBNF). We hypothesized (1) higher %NBNF coinciding with higher P supply, and (2) higher %NBNF in mycorrhizal as compared to non-mycorrhizal plants under P deficiency due to mycorrhiza-mediated improvement in P nutrition. We found a strongly positive correlation between total plant P content and %NBNF, clearly documenting the importance of plant P nutrition for BNF efficiency. The AM symbiosis generally improved P uptake by plants and considerably stimulated the efficiency of BNF under low P availability (below 10 mg kg-1 water extractable P). Under high P availability (above 10 mg kg-1 water extractable P), the AM symbiosis brought no further benefits to the plants with respect to P nutrition even as the effects of P availability on N acquisition via BNF were further modulated by the environmental context (plant and substrate combinations). As a response to elevated P availability in the substrate, the extent of root length colonization by AM fungi was reduced, the turning points occurring at about 8 and 10 mg kg-1 water extractable P for M. sativa and M. truncatula, respectively. Our results indicated competition for limited C resource between the two kinds of microsymbionts and thus degradation of AM symbiotic functioning under ample P supply.