Biosynthesis of polyhydroxybutyrate by Methylorubrum extorquens DSM13060 is essential for intracellular colonization in plant endosymbiosis.

Methylorubrum extorquens DSM13060 is an endosymbiont that lives in the cells of shoot tip meristems. The bacterium is methylotrophic and consumes plant-derived methanol for the production of polyhydroxybutyrate (PHB). The PHB provides protection against oxidative stress for both host and endosymbiont cells through its fragments, methyl-esterified 3-hydroxybutyrate (ME-3HB) oligomers. We evaluated the role of the genes involved in the production of ME-3HB oligomers in the host colonization by the endosymbiont M. extorquens DSM13060 through targeted genetic mutations. The strains with deletions in PHB synthase (phaC), PHB depolymerase (phaZ1), and a transcription factor (phaR) showed altered PHB granule characteristics, as ΔphaC had a significantly low number of granules, ΔphaR had a significantly increased number of granules, and ΔphaZ1 had significantly large PHB granules in the bacterial cells. When the deletion strains were exposed to oxidative stress, the ΔphaC strain was sensitive to 10 mM HO· and 20 mM H2O2. The colonization of the host, Scots pine (Pinus sylvestris L.), by the deletion strains varied greatly. The deletion strain ΔphaR colonized the host mainly intercellularly, whereas the ΔphaZ1 strain was a slightly poorer colonizer than the control. The deletion strain ΔphaC lacked the colonization potential, living mainly on the surfaces of the epidermis of pine roots and shoots in contrast to the control, which intracellularly colonized all pine tissues within the study period. In earlier studies, deletions within the PHB metabolic pathway have had a minor effect on plant colonization by rhizobia. We have previously shown the association between ME-3HB oligomers, produced by PhaC and PhaZ1, and the ability to alleviate host-generated oxidative stress during plant infection by the endosymbiont M. extorquens DSM13060. Our current results show that the low capacity for PHB synthesis leads to poor tolerance of oxidative stress and loss of colonization potential by the endosymbiont. Altogether, our findings demonstrate that the metabolism of PHB in M. extorquens DSM13060 is an important trait in the non-rhizobial endosymbiosis.

Weather in two climatic regions shapes the diversity and drives the structure of fungal endophytic community of bilberry (Vaccinium myrtillus L.) fruit.

BACKGROUND: Bilberry (Vaccinium myrtillus L.) is one of the most important economic and natural resources in Northern Europe. Despite its importance, the endophytic fungal community of the fruits has rarely been investigated. Biogeographic patterns and determinants of the fungal diversity in the bilberry fruit are poorly understood, albeit fungal endophytes can have a close relationship with the host plants. Here, we investigated the effect of climatic regions, and their weather conditions within growth season and soil properties on fungal endophytic communities of bilberry fruits collected from northern and southern regions of Finland using high-throughput sequencing technology targeting the internal transcribed spacer 2 ribosomal DNA region for fungi. RESULTS: Species richness and beta diversity (variation in community structure) were higher in the southern compared to the studied northern region. The weather condition of the growth season drove both fungal richness and community structure. Furthermore, abundance of the genera Venturia, Cladosporium, and Podosphaera was influenced by the weather, being different between the south and north regions. CONCLUSIONS: We conclude that diversity and assembly structure of the fungal endophytes in bilberry fruits follow similar patterns as for foliar fungal endophytes, being shaped by various environmental factors, such as the climate and surrounding vegetation.

The meristem-associated endosymbiont Methylorubrum extorquens DSM13060 reprograms development and stress responses of pine seedlings.

Microbes living in plant tissues-endophytes-are mainly studied in crop plants where they typically colonize the root apoplast. Trees-a large carbon source with a high capacity for photosynthesis-provide a variety of niches for endophytic colonization. We have earlier identified a new type of plant-endophyte interaction in buds of adult Scots pine, where Methylorubrum species live inside the meristematic cells. The endosymbiont Methylorubrum extorquens DSM13060 significantly increases needle and root growth of pine seedlings without producing plant hormones, but by aggregating around host nuclei. Here, we studied gene expression and metabolites of the pine host induced by M. extorquens DSM13060 infection. Malic acid was produced by pine to potentially boost M. extorquens colonization and interaction. Based on gene expression, the endosymbiont activated the auxin- and ethylene (ET)-associated hormonal pathways through induction of CUL1 and HYL1, and suppressed salicylic and abscisic acid signaling of pine. Infection by the endosymbiont had an effect on pine meristem and leaf development through activation of GLP1-7 and ALE2, and suppressed flowering, root hair and lateral root formation by downregulation of AGL8, plantacyanin, GASA7, COW1 and RALFL34. Despite of systemic infection of pine seedlings by the endosymbiont, the pine genes CUL1, ETR2, ERF3, HYL, GLP1-7 and CYP71 were highly expressed in the shoot apical meristem, rarely in needles and not in stem or root tissues. Low expression of MERI5, CLH2, EULS3 and high quantities of ononitol suggest that endosymbiont promotes viability and protects pine seedlings against abiotic stress. Our results indicate that the endosymbiont positively affects host development and stress tolerance through mechanisms previously unknown for endophytic bacteria, manipulation of plant hormone signaling pathways, downregulation of senescence and cell death-associated genes and induction of ononitol biosynthesis.

Host species shape the community structure of culturable endophytes in fruits of wild berry species (Vaccinium myrtillus L., Empetrum nigrum L. and Vaccinium vitis-idaea L.).

Wild berries are interesting research subjects due to their rich sources of health-beneficial phenolic compounds. However, the internal microbial communities, endophytes, associated with the wild berry fruits are currently unknown. Endophytes are bacteria or fungi inhabiting inside plant tissues, and their functions vary depending on the host species and environmental parameters. The present study aimed to examine community composition of fungal and bacterial endophytes in fruits of three wild berry species (bilberry Vaccinium myrtillus L., lingonberry Vaccinium vitis-idaea L. and crowberry Empetrum nigrum L.) and the effects of host plant species and their growth sites on shaping the endophytic communities. We found that the endophytic community structures differed between the berry species, and fungi were predominant over bacteria in the total endophytic taxa. We identified previously unknown endophytic fungal taxa including Angustimassarina, Dothidea, Fellozyma, Pseudohyphozyma, Hannaella coprosmae and Oberwinklerozyma straminea. A role of soluble phenolic compounds, the intracellular components in wild berry fruits, in shaping the endophytic communities is proposed. Overall, our study demonstrates that each berry species harbors a unique endophytic community of microbes.

Compensatory IgM to the Rescue: Patients with Selective IgA Deficiency Have Increased Natural IgM Antibodies to MAA-LDL and No Changes in Oral Microbiota.

IgA is the most abundant Ab in the human body. However, most patients with selective IgA deficiency (SIgAD) are asymptomatic. IgM, and to lesser extent IgG Abs, are generally presumed to compensate for the lack of IgA in SIgAD by multiplying and adopting functions of IgA. We used data from the Northern Finland Birth Cohort 1966 to investigate whether SIgAD patients have differences in levels of natural Abs to oxidized epitopes compared with 20 randomly selected healthy controls. First, we screened the saliva and serum samples from the Northern Finland Birth Cohort 1966 cohort (n = 1610) for IgA concentration. We detected five IgA-deficient subjects, yielding a prevalence of 0.3%, which is consistent with the general prevalence of 0.25% in the Finnish population. To detect natural Abs, we used malondialdehyde acetaldehyde-low-density lipoprotein (MAA-LDL), an Ag known to bind natural Abs. In this study, we show that natural secretory IgM and IgG Abs to MAA-DL were significantly increased in subjects with SIgAD. Given that secretory IgA is an important part of mucosal immune defense and that, in the gut microbiota, dysbiosis with SIgAD patients has been observed, we characterized the oral bacterial microbiota of the subjects with and without SIgAD using high-throughput 16S rRNA gene sequencing. We found no significant alterations in diversity and composition of the oral microbiota in subjects with SIgAD. Our data suggest that increased levels of secretory natural Abs in patients with SIgAD could be a compensatory mechanism, providing alternative first-line defense against infections and adjusting mucosal milieu to maintain a healthy oral microbiota.

Biofertilizers and Biocontrol Agents for Agriculture: How to Identify and Develop New Potent Microbial Strains and Traits.

Microbiological tools, biofertilizers, and biocontrol agents, which are bacteria and fungi capable of providing beneficial outcomes in crop plant growth and health, have been developed for several decades. Currently we have a selection of strains available as products for agriculture, predominantly based on plant-growth-promoting rhizobacteria (PGPR), soil, epiphytic, and mycorrhizal fungi, each having specific challenges in their production and use, with the main one being inconsistency of field performance. With the growing global concern about pollution, greenhouse gas accumulation, and increased need for plant-based foods, the demand for biofertilizers and biocontrol agents is expected to grow. What are the prospects of finding solutions to the challenges on existing tools? The inconsistent field performance could be overcome by using combinations of several different types of microbial strains, consisting various members of the full plant microbiome. However, a thorough understanding of each microbiological tool, microbial communities, and their mechanisms of action must precede the product development. In this review, we offer a brief overview of the available tools and consider various techniques and approaches that can produce information on new beneficial traits in biofertilizer and biocontrol strains. We also discuss innovative ideas on how and where to identify efficient new members for the biofertilizer and biocontrol strain family.

Does Intraspecific Variation in rDNA Copy Number Affect Analysis of Microbial Communities?

Amplicon sequencing of partial regions of the ribosomal RNA loci (rDNA) is widely used to profile microbial communities. However, the rDNA is dynamic and can exhibit substantial interspecific and intraspecific variation in copy number in prokaryotes and, especially, in microbial eukaryotes. As change in rDNA copy number is a common response to environmental change, rDNA copy number is not necessarily a property of a species. Variation in rDNA copy number, especially the capacity for large intraspecific changes driven by external cues, complicates analyses of rDNA amplicon sequence data. We highlight the need to (i) interpret amplicon sequence data in light of possible interspecific and intraspecific variation, and (ii) examine the potential plasticity in rDNA copy number as an important ecological factor to better understand how microbial communities are structured in heterogeneous environments.

The protective role of PHB and its degradation products against stress situations in bacteria.

Many bacteria produce storage biopolymers that are mobilized under conditions of metabolic adaptation, for example, low nutrient availability and cellular stress. Polyhydroxyalkanoates are often found as carbon storage in Bacteria or Archaea, and of these polyhydroxybutyrate (PHB) is the most frequently occurring PHA type. Bacteria usually produce PHB upon availability of a carbon source and limitation of another essential nutrient. Therefore, it is widely believed that the function of PHB is to serve as a mobilizable carbon repository when bacteria face carbon limitation, supporting their survival. However, recent findings indicate that bacteria switch from PHB synthesis to mobilization under stress conditions such as thermal and oxidative shock. The mobilization products, 3-hydroxybutyrate and its oligomers, show a protective effect against protein aggregation and cellular damage caused by reactive oxygen species and heat shock. Thus, bacteria should have an environmental monitoring mechanism directly connected to the regulation of the PHB metabolism. Here, we review the current knowledge on PHB physiology together with a summary of recent findings on novel functions of PHB in stress resistance. Potential applications of these new functions are also presented.

Fungal Dysbiosis and Intestinal Inflammation in Children With Beta-Cell Autoimmunity.

Although gut bacterial dysbiosis is recognized as a regulator of beta-cell autoimmunity, no data is available on fungal dysbiosis in the children at the risk of type 1 diabetes (T1D). We hypothesized that the co-occurrence of fungal and bacterial dysbiosis contributes to the intestinal inflammation and autoimmune destruction of insulin-producing beta-cells in T1D. Fecal and blood samples were collected from 26 children tested positive for at least one diabetes-associated autoantibody (IAA, GADA, IA-2A or ICA) and matched autoantibody-negative children with HLA-conferred susceptibility to T1D (matched for HLA-DQB1 haplotype, age, gender and early childhood nutrition). Bacterial 16S and fungal ITS2 sequencing, and analyses of the markers of intestinal inflammation, namely fecal human beta-defensin-2 (HBD2), calprotectin and secretory total IgA, were performed. Anti-Saccharomyces cerevisiae antibodies (ASCA) and circulating cytokines, IFNG, IL-17 and IL-22, were studied. After these analyses, the children were followed for development of clinical T1D (median 8 years and 8 months). Nine autoantibody positive children were diagnosed with T1D, whereas none of the autoantibody negative children developed T1D during the follow-up. Fungal dysbiosis, characterized by high abundance of fecal Saccharomyces and Candida, was found in the progressors, i.e., children with beta-cell autoimmunity who during the follow-up progressed to clinical T1D. These children showed also bacterial dysbiosis, i.e., increased Bacteroidales and Clostridiales ratio, which was, however, found also in the non-progressors, and is thus a common nominator in the children with beta-cell autoimmunity. Furthermore, the progressors showed markers of intestinal inflammation detected as increased levels of fecal HBD2 and ASCA IgG to fungal antigens. We conclude that the fungal and bacterial dysbiosis, and intestinal inflammation are associated with the development of T1D in children with beta-cell autoimmunity.

Corpse management of the invasive Argentine ant inhibits growth of pathogenic fungi.

A dead conspecific poses a potential pathogen risk for social animals. We have discovered that Argentine ants (Linepithema humile) prevent spread of pathogenic fungi from corpses by depositing the dead to combined toilet and refuse areas and applying pygidial gland secretion on them. The presence of a corpse in a nest increases this secretion behaviour. We identified three fungi growing on Argentine ant corpses. Growth of the Argentine ant pathogen Aspergillus nomius and the plant pathogen Fusarium solani on corpses was inhibited as long as the ants were constantly attending them as the ant anal secretion only delayed germination of their spores. In contrast, the effect of the ant anal secretion on the human pathogen Aspergillus fumigatus was much stronger: it prevented spore germination and, accordingly, the fungus no longer grew on the treated corpses. The Argentine ants are one of the world's worst invasive alien species as they cause ecological and economical damage in their new habitats. Our discovery points at a novel method to limit Argentine ant colonies through their natural fungal pathogens.

Different endophyte communities colonize buds of sprouts compared with mature trees of mountain birch recovered from moth herbivory.

Plant meristems were previously thought to be sterile. Today, meristem-associated shoot endophytes are mainly reported as contaminants from plant tissue cultures, the number of observed species being very low. However, the few strains characterized have the capacity for infecting host cells and affecting plant growth and development. Here we studied the communities of endophytic bacteria in the buds of mountain birch (Betula pubescens ssp. czerepanovii (N. I. Orlova) Hämet-Ahti) exposed to winter moth (Operophtera brumata L.) herbivory, to identify differences between sprouts and branches of mature birch trees. Mountain birch of the high subarctic is cyclically exposed to winter moth and produces sprouts to generate new trees as a survival mechanism. The majority (54%) of operational taxonomic units belonged to Xanthomonadaceae and Pseudomonales of Proteobacteria. Most of the observed species were classified as Xanthomonas (28%). Sprout buds had the highest diversity, containing approximately three times more species, and significantly more (43%) Pseudomonas species than the mature trees (14%). Our results demonstrate that endophytic communities of buds are richer than previously thought. We suggest that the meristem-associated endophytes should be studied further for a possible role in sprouting and aiding regeneration of trees.

Methyl-esterified 3-hydroxybutyrate oligomers protect bacteria from hydroxyl radicals.

Bacteria rely mainly on enzymes, glutathione and other low-molecular weight thiols to overcome oxidative stress. However, hydroxyl radicals are the most cytotoxic reactive oxygen species, and no known enzymatic system exists for their detoxification. We now show that methyl-esterified dimers and trimers of 3-hydroxybutyrate (ME-3HB), produced by bacteria capable of polyhydroxybutyrate biosynthesis, have 3-fold greater hydroxyl radical-scavenging activity than glutathione and 11-fold higher activity than vitamin C or the monomer 3-hydroxybutyric acid. We found that ME-3HB oligomers protect hypersensitive yeast deletion mutants lacking oxidative stress-response genes from hydroxyl radical stress. Our results show that phaC and phaZ, encoding polymerase and depolymerase, respectively, are activated and polyhydroxybutyrate reserves are degraded for production of ME-3HB oligomers in bacteria infecting plant cells and exposed to hydroxyl radical stress. We found that ME-3HB oligomer production is widespread, especially in bacteria adapted to stressful environments. We discuss how ME-3HB oligomers could provide opportunities for numerous applications in human health.

The intracellular Scots pine shoot symbiont Methylobacterium extorquens DSM13060 aggregates around the host nucleus and encodes eukaryote-like proteins.

UNLABELLED: Endophytes are microbes that inhabit plant tissues without any apparent signs of infection, often fundamentally altering plant phenotypes. While endophytes are typically studied in plant roots, where they colonize the apoplast or dead cells, Methylobacterium extorquens strain DSM13060 is a facultatively intracellular symbiont of the meristematic cells of Scots pine (Pinus sylvestris L.) shoot tips. The bacterium promotes host growth and development without the production of known plant growth-stimulating factors. Our objective was to examine intracellular colonization by M. extorquens DSM13060 of Scots pine and sequence its genome to identify novel molecular mechanisms potentially involved in intracellular colonization and plant growth promotion. Reporter construct analysis of known growth promotion genes demonstrated that these were only weakly active inside the plant or not expressed at all. We found that bacterial cells accumulate near the nucleus in intact, living pine cells, pointing to host nuclear processes as the target of the symbiont's activity. Genome analysis identified a set of eukaryote-like functions that are common as effectors in intracellular bacterial pathogens, supporting the notion of intracellular bacterial activity. These include ankyrin repeats, transcription factors, and host-defense silencing functions and may be secreted by a recently imported type IV secretion system. Potential factors involved in host growth include three copies of phospholipase A2, an enzyme that is rare in bacteria but implicated in a range of plant cellular processes, and proteins putatively involved in gibberellin biosynthesis. Our results describe a novel endophytic niche and create a foundation for postgenomic studies of a symbiosis with potential applications in forestry and agriculture. IMPORTANCE: All multicellular eukaryotes host communities of essential microbes, but most of these interactions are still poorly understood. In plants, bacterial endophytes are found inside all tissues. M. extorquens DSM13060 occupies an unusual niche inside cells of the dividing shoot tissues of a pine and stimulates seedling growth without producing cytokinin, auxin, or other plant hormones commonly synthesized by plant-associated bacteria. Here, we tracked the bacteria using a fluorescent tag and confocal laser scanning microscopy and found that they localize near the nucleus of the plant cell. This prompted us to sequence the genome and identify proteins that may affect host growth by targeting processes in the host cytoplasm and nucleus. We found many novel genes whose products may modulate plant processes from within the plant cell. Our results open up new avenues to better understand how bacteria assist in plant growth, with broad implications for plant science, forestry, and agriculture.

Interaction with ectomycorrhizal fungi and endophytic Methylobacterium affects nutrient uptake and growth of pine seedlings in vitro.

Tissues of Scots pine (Pinus sylvestris L.) contain several endophytic microorganisms of which Methylobacterium extorquens DSM13060 is a dominant species throughout the year. Similar to other endophytic bacteria, M. extorquens is able to colonize host plant tissues without causing any symptoms of disease. In addition to endophytic bacteria, plants associate simultaneously with a diverse set of microorganisms. Furthermore, plant-colonizing microorganisms interact with each other in a species- or strain-specific manner. Several studies on beneficial microorganisms interacting with plants have been carried out, but few deal with interactions between different symbiotic organisms and specifically, how these interactions affect the growth and development of the host plant. Our aim was to study how the pine endophyte M. extorquens DSM13060 affects pine seedlings and how the co-inoculation with ectomycorrhizal (ECM) fungi [Suillus variegatus (SV) or Pisolithus tinctorius (PT)] alters the response of Scots pine. We determined the growth, polyamine and nutrient contents of inoculated and non-inoculated Scots pine seedlings in vitro. Our results show that M. extorquens is able to improve the growth of seedlings at the same level as the ECM fungi SV and PT do. The effect of co-inoculation using different symbiotic organisms was seen in terms of changes in growth and nutrient uptake. Inoculation using M. extorquens together with ECM fungi improved the growth of the host plant even more than single ECM inoculation. Symbiotic organisms also had a strong effect on the potassium content of the seedling. The results indicate that interaction between endophyte and ECM fungus is species dependent, leading to increased or decreased nutrient content and growth of pine seedlings.

Localization of strawberry (Fragaria x ananassa) and Methylobacterium extorquens genes of strawberry flavor biosynthesis in strawberry tissue by in situ hybridization.

Strawberry flavor is one of the most popular fruit flavors worldwide, with numerous applications in the food industry. In addition, the biosynthetic origin of the most important strawberry flavor components, such as 2,5-dimethyl-4-hydroxy-2H-furan-3-one (DMHF), is a challenging research area. DMHF's precursor, 2-hydroxy-propanal (or lactaldehyde), is biosynthesized by the endophytic bacterium Methylobacterium extorquens (M. extorquens). In particular, the alcohol dehydrogenase (ADH) enzymes of M. extorquens are involved in the biogenesis of DMHF precursors since they have the capacity to oxidize the strawberry-derived 1,2-propanediol to lactaldehyde. In this study, the expression of the endophytic ADH and the plant DMHF biosynthesis genes was examined in the tissues of raw and ripe strawberry receptacles by in situ hybridization. The presence of endophytic bacteria was studied in the same tissues by probes targeting bacterial 16S ribosomal ribonucleic acid. Hybridization signals of probes specific for endophytic ADH and plant DMHF biosynthesis genes, as well as bacteria-specific probes, were detected in the same locations. The probes were localized near the plasma membranes or intercellular spaces of cortical and vascular tissues of the receptacle, and intracellularly in the tissues of achenes. By localizing the expression of the endophytic methanol ADH and plant DMHF biosynthesis genes to the same tissues, we have reinforced our original hypothesis that an intimate symbiotic relationship between strawberry and endophytic cells exists and leads to the biosynthesis of DMHF.

A SQUAMOSA MADS box gene involved in the regulation of anthocyanin accumulation in bilberry fruits.

Anthocyanins are important health-promoting phytochemicals that are abundant in many fleshy fruits. Bilberry (Vaccinium myrtillus) is one of the best sources of these compounds. Here, we report on the expression pattern and functional analysis of a SQUAMOSA-class MADS box transcription factor, VmTDR4, associated with anthocyanin biosynthesis in bilberry. Levels of VmTDR4 expression were spatially and temporally linked with color development and anthocyanin-related gene expression. Virus-induced gene silencing was used to suppress VmTDR4 expression in bilberry, resulting in substantial reduction in anthocyanin levels in fully ripe fruits. Chalcone synthase was used as a positive control in the virus-induced gene silencing experiments. Additionally, in sectors of fruit tissue in which the expression of the VmTDR4 gene was silenced, the expression of R2R3 MYB family transcription factors related to the biosynthesis of flavonoids was also altered. We conclude that VmTDR4 plays an important role in the accumulation of anthocyanins during normal ripening in bilberry, probably through direct or indirect control of transcription factors belonging to the R2R3 MYB family.

Mycobacteria are hidden endophytes in the shoots of rock plant [Pogonatherum paniceum (Lam.) Hack.] (Poaceae).

A mycobacterium was isolated from micropropagated Pogonatherum paniceum and identified as a close relative of Mycobacterium cookii. The endophyte diversity in the shoots of potted and micropropagated P. paniceum plants was studied by culture-independent techniques. Group- and strain-specific PCR demonstrated that the P. paniceum plants harboured the isolated Mycobacterium strain as a minority. Altogether 101 clones of the PCR products were sequenced. The shoots of potted P. paniceum plants harboured unculturable endophytes in the families Phyllobacteriaceae, Hyphomicrobiaceae, Sphingobacteriaceae, Enterobacteriaceae, Alcaligenaceae and Mycobacteriaceae. Among the unculturable Mycobacteriaceae strains related to Mycobacterium chubuense, M. poriferae, M. obuense, M. fortuitum, M. neoaurum, M. diernhoferi, M. intracellulare and M. cookii were identified. Three unique sequences that clustered with M. llatzarense and M. mucogenicum were identified in micropropagated plants. According to the results, the shoots and micropropagated tissues of rock plant are inhabited by mycobacteria, which should stimulate further studies on the diversity of unculturable mycobacteria in edible crop plants.