Plant-endophyte communication: Scaling from molecular mechanisms to ecological outcomes.

Diverse communities of fungal endophytes reside in plant tissues, where they affect and are affected by plant physiology and ecology. For these intimate interactions to form and persist, endophytes and their host plants engage in intricate systems of communication. The conversation between fungal endophytes and plant hosts ultimately dictates endophyte community composition and function and has cascading effects on plant health and plant interactions. In this review, we synthesize our current knowledge on the mechanisms and strategies of communication used by endophytic fungi and their plant hosts. We discuss the molecular mechanisms of communication that lead to organ specificity of endophytic communities and distinguish endophytes, pathogens, and saprotrophs. We conclude by offering emerging perspectives on the relevance of plant-endophyte communication to microbial community ecology and plant health and function.

Epichloë Increases Root Fungal Endophyte Richness and Alters Root Fungal Endophyte Composition in a Changing World.

Plants harbor a variety of fungal symbionts both above- and belowground, yet little is known about how these fungi interact within hosts, especially in a world where resource availability is changing due to human activities. Systemic vertically transmitted endophytes such as Epichloë spp. may have particularly strong effects on the diversity and composition of later-colonizing symbionts such as root fungal endophytes, especially in primary successional systems. We made use of a long-term field experiment in Great Lakes sand dunes to test whether Epichloë colonization of the dune-building grass, Ammophila breviligulata, could alter fungal root endophyte species richness or community composition in host plants. We also tested whether nitrogen addition intensified the effects of Epichlöe on the root endophyte community. We found that Epichloë increased richness of root endophytes in Ammophila by 17% overall, but only shifted community composition of root endophytes under nitrogen-enriched conditions. These results indicate that Epichlöe acts as a key species within Ammophila, changing richness and composition of the root mycobiome and integrating above- and belowground mycobiome interactions. Further, effects of Epichloë on root endophyte communities were enhanced by N addition, indicating that this fungal species may become even more important in future environments.

Thyroid and Parathyroid Conditions: Hyperthyroidism.

Hyperthyroidism is an excess in thyroid hormone production caused by such conditions as Graves disease, toxic multinodular goiter, and toxic adenoma. Overt hyperthyroidism is defined as a low or undetectable thyrotropin (TSH) level with elevated triiodothyronine (T3) or thyroxine (T4) values, whereas subclinical hyperthyroidism is defined as low or undetectable TSH with normal T3 and T4 levels. Symptoms of hyperthyroidism include nervousness, heat intolerance, weight loss, and fatigue. The long-term consequences of unmanaged or poorly managed hyperthyroidism include increased risk of all-cause mortality, cardiovascular events, atrial fibrillation, sexual dysfunction, and osteoporosis. Overt and subclinical hyperthyroidism can be managed effectively with antithyroid drugs (eg, propylthiouracil, methimazole) or with definitive therapies (eg, radioactive iodine ablation, thyroidectomy). Subclinical hyperthyroidism is not always treated, although close monitoring is needed to prevent disease complications or progression to overt hyperthyroidism. Treatment for subclinical hyperthyroidism is recommended for patients 65 years or older with TSH levels lower than 0.10 mIU/L. Treatment also is recommended for symptomatic patients or those with cardiac or osteoporotic risk factors. Thyroid storm is a life-threatening complication of unmanaged or inadequately managed hyperthyroidism that warrants urgent treatment in a hospital setting.

Thyroid and Parathyroid Conditions: Hypothyroidism.

Hypothyroidism is caused by deficient thyroid hormone production secondary to autoimmune disease or insufficient iodine consumption or as a complication of hyperthyroidism management. Signs and symptoms include fatigue, weight gain, dry skin, constipation, and cold intolerance. The U.S. Preventive Services Task Force found insufficient evidence to recommend for or against screening for hypothyroidism, but some organizations support screening in special populations. If hypothyroidism is suspected, initial laboratory evaluation consists of a serum thyrotropin (TSH) measurement with reflex testing of free thyroxine (T4). Thyroid function tests must be interpreted carefully because acute illness, diet, and drugs may alter values. Overt hypothyroidism occurs when a patient has an elevated TSH level and a low free T4 level with symptoms of hypothyroidism. Management includes thyroid hormone replacement, ideally levothyroxine. Subclinical hypothyroidism is characterized by an elevated TSH level with a normal T4 value. The decision to treat subclinical hypothyroidism should be based on patient characteristics and shared decision-making discussions. Special consideration should be taken in treating patients with high-risk conditions, including heart disease, pregnancy, and myxedema coma, and in patients requiring high-dose levothyroxine. Thyroid hormone should be titrated based on goal TSH values, symptoms, and potential treatment adverse effects.

Thyroid and Parathyroid Conditions: Thyroid Nodules, Cysts, and Malignancies.

Thyroid nodules are identified incidentally on imaging in most patients. Controversy exists on which patients warrant evaluation of an incidental thyroid nodule. If further assessment of a nodule detected on imaging or examination is pursued, thyroid ultrasonography with cervical lymph node survey and measurement of serum thyrotropin (TSH) may guide management decisions. When the TSH level is low, a nuclear medicine thyroid scan is necessary. Based on size, ultrasonographic features, and nuclear medicine results, patients with thyroid nodules may undergo ultrasonographic surveillance or biopsy with fine-needle aspiration. When fine-needle aspiration is performed, the Bethesda System for Reporting Thyroid Cytopathology (BSRTC) provides a classification system for biopsy results. Molecular testing can be considered in the case of nodules with indeterminate findings based on biopsy. Malignant thyroid nodules and indeterminate nodules with suspicious molecular test results warrant surgical evaluation, whereas others may be monitored with periodic ultrasonography. Approximately 10% of nodules are clinically significant malignancies, and a small number of nodules cause compressive symptoms or progress to functional thyroid disease. Thyroid cancer overall has a 5-year survival of 98%.

Elevated carbon dioxide reduces a common soybean leaf endophyte.

Free-air CO2 enrichment (FACE) experiments have elucidated how climate change affects plant physiology and production. However, we lack a predictive understanding of how climate change alters interactions between plants and endophytes, critical microbial mediators of plant physiology and ecology. We leveraged the SoyFACE facility to examine how elevated [CO2 ] affected soybean (Glycine max) leaf endophyte communities in the field. Endophyte community composition changed under elevated [CO2 ], including a decrease in the abundance of a common endophyte, Methylobacterium sp. Moreover, Methylobacterium abundance was negatively correlated with co-occurring fungal endophytes. We then assessed how Methylobacterium affected the growth of co-occurring endophytic fungi in vitro. Methylobacterium antagonized most co-occurring fungal endophytes in vitro, particularly when it was more established in culture before fungal introduction. Variation in fungal response to Methylobacterium within a single fungal operational taxonomic unit (OTU) was comparable to inter-OTU variation. Finally, fungi isolated from elevated vs. ambient [CO2 ] plots differed in colony growth and response to Methylobacterium, suggesting that increasing [CO2 ] may affect fungal traits and interactions within the microbiome. By combining in situ and in vitro studies, we show that elevated [CO2 ] decreases the abundance of a common bacterial endophyte that interacts strongly with co-occurring fungal endophytes. We suggest that endophyte responses to global climate change will have important but largely unexplored implications for both agricultural and natural systems.

Host affinity of endophytic fungi and the potential for reciprocal interactions involving host secondary chemistry.

PREMISE: Interactions between fungal endophytes and their host plants present useful systems for identifying important factors affecting assembly of host-associated microbiomes. Here we investigated the role of secondary chemistry in mediating host affinity of asymptomatic foliar endophytic fungi using Psychotria spp. and Theobroma cacao (cacao) as hosts. METHODS: First, we surveyed endophytic communities in Psychotria species in a natural common garden using culture-based methods. Then we compared differences in endophytic community composition with differences in foliar secondary chemistry in the same host species, determined by liquid chromatography-tandem mass spectrometry. Finally, we tested how inoculation with live and heat-killed endophytes affected the cacao chemical profile. RESULTS: Despite sharing a common environment and source pool for endophyte spores, different Psychotria host species harbored strikingly different endophytic communities that reflected intrinsic differences in their leaf chemical profiles. In T. cacao, inoculation with live and heat-killed endophytes produced distinct cacao chemical profiles not found in uninoculated plants or pure fungal cultures, suggesting that endophytes, like pathogens, induce changes in secondary chemical profiles of their host plant. CONCLUSIONS: Collectively our results suggest at least two potential processes: (1) Plant secondary chemistry influences assembly and composition of fungal endophytic communities, and (2) host colonization by endophytes subsequently induces changes in the host chemical landscape. We propose a series of testable predictions based on the possibility that reciprocal chemical interactions are a general property of plant-endophyte interactions.

Foliar fungal endophyte community structure is independent of phylogenetic relatedness in an Asteraceae common garden.

Phylogenetic distance among host species represents a proxy for host traits that act as biotic filters to shape host-associated microbiome community structure. However, teasing apart potential biotic assembly mechanisms, such as host specificity or local species interactions, from abiotic factors, such as environmental specificity or dispersal barriers, in hyperdiverse, horizontally transmitted microbiomes remains a challenge. In this study, we tested whether host phylogenetic relatedness among 18 native Asteraceae plant species and spatial distance between replicated plots in a common garden affects foliar fungal endophyte (FFE) community structure. We found that FFE community structure varied significantly among host species, as well as host tribes, but not among host subfamilies. However, FFE community dissimilarity between host individuals was not significantly correlated with phylogenetic distance between host species. There was a significant effect of spatial distance among host individuals on FFE community dissimilarity within the common garden. The significant differences in FFE community structure among host species, but lack of a significant host phylogenetic effect, suggest functional differences among host species not accounted for by host phylogenetic distance, such as metabolic traits or phenology, may drive FFE community dissimilarity. Overall, our results indicate that host species identity and the spatial distance between plants can determine the similarity of their microbiomes, even across a single experimental field, but that host phylogeny is not closely tied to FFE community divergence in native Asteraceae.

Fungal functional ecology: bringing a trait-based approach to plant-associated fungi.

Fungi play many essential roles in ecosystems. They facilitate plant access to nutrients and water, serve as decay agents that cycle carbon and nutrients through the soil, water and atmosphere, and are major regulators of macro-organismal populations. Although technological advances are improving the detection and identification of fungi, there still exist key gaps in our ecological knowledge of this kingdom, especially related to function. Trait-based approaches have been instrumental in strengthening our understanding of plant functional ecology and, as such, provide excellent models for deepening our understanding of fungal functional ecology in ways that complement insights gained from traditional and -omics-based techniques. In this review, we synthesize current knowledge of fungal functional ecology, taxonomy and systematics and introduce a novel database of fungal functional traits (FunFun ). FunFun is built to interface with other databases to explore and predict how fungal functional diversity varies by taxonomy, guild, and other evolutionary or ecological grouping variables. To highlight how a quantitative trait-based approach can provide new insights, we describe multiple targeted examples and end by suggesting next steps in the rapidly growing field of fungal functional ecology.

Foliar endophytic fungi alter patterns of nitrogen uptake and distribution in Theobroma cacao.

Colonization by foliar endophytic fungi can affect the expression of host plant defenses and other ecologically important traits. However, whether endophyte colonization affects the uptake or redistribution of resources within and among host plant tissues remains unstudied. We inoculated leaves of Theobroma cacao with four common colonizers that range in their effect from protective to pathogenic (Colletotrichum tropicale, Pestalotiopsis sp., Colletotrichum theobromicola, or Phytophthora palmivora). We pulsed the soil with nitrogen-15 (15 N) and then traced 15 N uptake and its subsequent distribution to whole plants and individual leaves. At a whole-plant level, C. tropicale-inoculated plants showed significantly greater 15 N uptake than endophyte-free plants did in the same pot. Among leaves within plants, younger leaves were particularly enriched in 15 N, but endophyte inoculation at the individual leaf level did not alter 15 N distribution within plants. However, leaves co-inoculated with pathogenic Phytophthora and protective C. tropicale experienced significantly elevated 15 N content as pathogen damage increased, compared with leaves inoculated only with the pathogen. Further, endophyte-pathogen co-infection also increased total plant biomass. Our results indicate that colonization by foliar endophytes significantly affects N uptake and distribution among and within host plants in ways that appear to be context dependent on other microbiome components.

Using Scaffolding and Deliberate Practice to Improve Abstract Writing in an Introductory Biology Laboratory Course.

s play the pivotal role of selling an article to a prospective reader, and for students, the ability to communicate science in concise written form may foster scientific thinking. However, students struggle with abstract composition, and we lack evidence-based educational innovations to help them develop this skill. We designed, implemented, and assessed an intervention for abstract composition with elements of scaffolding and transparency to ask whether deliberate practice improves concise scientific writing in early career undergraduate biology majors. We evaluated student performance by analyzing abstracts written before and after the intervention and by assessing pre- and posttest student concept maps. We found that scaffolded learning improved student abstract writing, with the greatest gains in students' ability to describe the motivation for their work. Using a set of tested tools to teach scientific writing has important implications for strengthening students' capacity to reinforce and synthesize content in the future, whether that is in laboratory course exercises, in independent research, or as a transferable skill to general critical thinking.

Carbon allocation and competition maintain variation in plant root mutualisms.

Plants engage in multiple root symbioses that offer varying degrees of benefit. We asked how variation in partner quality persists using a resource-ratio model of population growth. We considered the plant's ability to preferentially allocate carbon to mutualists and competition for plant carbon between mutualist and nonmutualist symbionts. We treated carbon as two nutritionally interchangeable, but temporally separated, resources-carbon allocated indiscriminately for the construction of the symbiosis, and carbon preferentially allocated to the mutualist after symbiosis establishment and assessment. This approach demonstrated that coexistence of mutualists and nonmutualists is possible when fidelity of the plant to the mutualist and the cost of mutualism mediate resource competition. Furthermore, it allowed us to trace symbiont population dynamics given varying degrees of carbon allocation. Specifically, coexistence occurs at intermediate levels of preferential allocation. Our findings are consistent with previous empirical studies as well the application of biological market theory to plantroot symbioses.

Exposure to the leaf litter microbiome of healthy adults protects seedlings from pathogen damage.

It is increasingly recognized that microbiota affect host health and physiology. However, it is unclear what factors shape microbiome community assembly in nature, and how microbiome assembly can be manipulated to improve host health. All plant leaves host foliar endophytic fungi, which make up a diverse, environmentally acquired fungal microbiota. Here, we experimentally manipulated assembly of the cacao tree (Theobroma cacao) fungal microbiome in nature and tested the effect of assembly outcome on host health. Using next-generation sequencing, as well as culture-based methods coupled with Sanger sequencing, we found that manipulating leaf litter exposure and location within the forest canopy significantly altered microbiome composition in cacao. Exposing cacao seedlings to leaf litter from healthy conspecific adults enriched the seedling microbiome with Colletotrichum tropicale, a fungal endophyte known to enhance pathogen resistance of cacao seedlings by upregulating host defensive pathways. As a result, seedlings exposed to healthy conspecific litter experienced reduced pathogen damage. Our results link processes that affect the assembly and composition of microbiome communities to their functional consequences for host success, and have broad implications for understanding plant-microbe interactions. Deliberate manipulation of the plant-fungal microbiome also has potentially important applications for cacao production and other agricultural systems in general.

Plant Host and Geographic Location Drive Endophyte Community Composition in the Face of Perturbation.

All plants form symbioses with endophytic fungi, which affect host plant health and function. Most endophytic fungi are horizontally transmitted, and consequently, local environment and geographic location greatly influence endophyte community composition. Growing evidence also suggests that identity of the plant host (e.g., species, genotype) can be important in shaping endophyte communities. However, little is known about how disturbances to plants affect their fungal symbiont communities. The goal of this study was to test if disturbances, from both natural and anthropogenic sources, can alter endophyte communities independent of geographic location or plant host identity. Using the plant species white snakeroot (Ageratina altissima; Asteraceae), we conducted two experiments that tested the effect of perturbation on endophyte communities. First, we examined endophyte response to leaf mining insect activity, a natural perturbation, in three replicate populations. Second, for one population, we applied fungicide to plant leaves to test endophyte community response to an anthropogenic perturbation. Using culture-based methods and Sanger sequencing of fungal isolates, we then examined abundance, diversity, and community structure of endophytic fungi in leaves subjected to perturbations by leaf mining and fungicide application. Our results show that plant host individual and geographic location are the major determinants of endophyte community composition even in the face of perturbations. Unexpectedly, we found that leaf mining did not impact endophyte communities in white snakeroot, but fungicide treatment resulted in small but significant changes in endophyte community structure. Together, our results suggest that endophyte communities are highly resistant to biotic and anthropogenic disturbances.

Microbiomes: unifying animal and plant systems through the lens of community ecology theory.

The field of microbiome research is arguably one of the fastest growing in biology. Bacteria feature prominently in studies on animal health, but fungi appear to be the more prominent functional symbionts for plants. Despite the similarities in the ecological organization and evolutionary importance of animal-bacterial and plant-fungal microbiomes, there is a general failure across disciplines to integrate the advances made in each system. Researchers studying bacterial symbionts in animals benefit from greater access to efficient sequencing pipelines and taxonomic reference databases, perhaps due to high medical and veterinary interest. However, researchers studying plant-fungal symbionts benefit from the relative tractability of fungi under laboratory conditions and ease of cultivation. Thus each system has strengths to offer, but both suffer from the lack of a common conceptual framework. We argue that community ecology best illuminates complex species interactions across space and time. In this synthesis we compare and contrast the animal-bacterial and plant-fungal microbiomes using six core theories in community ecology (i.e., succession, community assembly, metacommunities, multi-trophic interactions, disturbance, restoration). The examples and questions raised are meant to spark discussion amongst biologists and lead to the integration of these two systems, as well as more informative, manipulatory experiments on microbiomes research.

Medical school research intensity and academic rank of family medicine faculty.

BACKGROUND: Family medicine research productivity has been reported to be less than that of other academic disciplines. This difference could be reflected in academic success for family medicine faculty at schools where National Institutes of Health (NIH) funding in emphasized. This study sought to determine if academic rank for family medicine faculty was different at schools with higher NIH funding compared to those with less funding. METHODS: From the latest available list of NIH funding by institution, we selected 10 medical schools each from the higher, middle, and lowest tertile of overall funding (30 total schools). Using departmental web sites, we compared the percentage of family medicine faculty at assistant, associate, and professor rankings with those of other generalist divisions and two other sample departments. RESULTS: The distribution of faculty ranks for family medicine faculty does not differ based on the level of NIH funding of the school. When compared to other disciplines, family medicine faculty were less likely to be at advanced ranks compared to some disciplines (neurology) but more likely than in others (general internal medicine). CONCLUSION: The level of NIH funding does not appear to be a predictor of success at achieving higher faculty ranks for family medicine faculty.

In vivo dendritic cell tracking using fluorescence lifetime imaging and near-infrared-emissive polymersomes.

PURPOSE: Noninvasive in vivo cell-tracking techniques are necessary to advance the field of cellular-based therapeutics as well as to elucidate mechanisms governing in vivo cell biology. Fluorescence is commonly used for in vitro and postmortem biomedical studies but has been limited by autofluorescence at the whole-animal level. PROCEDURES: In this report, we demonstrate the ability of in vivo fluorescent lifetime imaging to remove autofluorescence and thereby enable in vivo dendritic cell tracking in naïve mice. Specifically, we track mature dendritic cells (DCs) labeled internally with near-infrared-emissive polymersomes (NIR-DCs). RESULTS: We establish the ability to detect labeled cells in vivo and image NIR-DC trafficking after both intravenous and subcutaneous delivery. In addition, we demonstrate the longitudinal capacity of this method by characterizing NIR-DC migration kinetics in the popliteal lymph node. CONCLUSIONS: This work provides a tool to evaluate dendritic-cell-based immunotherapy and generates novel opportunities for in vivo fluorescence imaging.

Genetic variation in the urea cycle: a model resource for investigating key candidate genes for common diseases.

The urea cycle is the primary means of nitrogen metabolism in humans and other ureotelic organisms. There are five key enzymes in the urea cycle: carbamoyl-phosphate synthetase 1 (CPS1), ornithine transcarbamylase (OTC), argininosuccinate synthetase (ASS1), argininosuccinate lyase (ASL), and arginase 1 (ARG1). Additionally, a sixth enzyme, N-acetylglutamate synthase (NAGS), is critical for urea cycle function, providing CPS1 with its necessary cofactor. Deficiencies in any of these enzymes result in elevated blood ammonia concentrations, which can have detrimental effects, including central nervous system dysfunction, brain damage, coma, and death. Functional variants, which confer susceptibility for disease or dysfunction, have been described for enzymes within the cycle; however, a comprehensive screen of all the urea cycle enzymes has not been performed. We examined the exons and intron/exon boundaries of the five key urea cycle enzymes, NAGS, and two solute carrier transporter genes (SLC25A13 and SLC25A15) for sequence alterations using single-stranded conformational polymorphism (SSCP) analysis and high-resolution melt profiling. SSCP was performed on a set of DNA from 47 unrelated North American individuals with a mixture of ethnic backgrounds. High-resolution melt profiling was performed on a nonoverlapping DNA set of either 47 or 100 unrelated individuals with a mixture of backgrounds. We identified 33 unarchived polymorphisms in this screen that potentially play a role in the variation observed in urea cycle function. Screening all the genes in the pathway provides a catalog of variants that can be used in investigating candidate diseases.

Tat-functionalized near-infrared emissive polymersomes for dendritic cell labeling.

Dendritic cells (DCs) play a pivotal role in both immune tolerance and the initiation of immunological responses. The ability to track DCs in vivo is imperative for the development of DC-based cellular therapies and to advance our understanding of DC function and pathophysiology. Here, we conjugate a cell permeable peptide, Tat, to near-infrared (NIR) emissive polymersomes in order to enable efficient intracellular delivery for future DC tracking with these optical probes. NIR imaging allows quantitative, repetitive, in vivo detection of fluorophore-laden cells, at centimeter tissue depths without disturbing cellular function. Flow cytometry and confocal microscopy results indicate that Tat-mediated polymersome delivery to DCs is concentration and time dependent, resulting in punctate intracellular localization. Further, loading cells with Tat NIR emissive polymersomes does not interfere with cytokine-induced DC maturation and has modest effects on DC viability, but has a significant effect on mature DC-induced activation of naive T cells. We observe significant uptake of NIR emissive polymersomes when conjugated to the peptide, with a lower detection limit of 5000 labeled DCs. The extent of polymersome delivery is estimated as 70 000 +/- 10 000 vesicles/cell, equivalent to 0.7 +/- 0.1 fmol of NIR fluorophore. Our studies will enable future in vivo tracking of ex vivo labeled DCs by NIR fluorescence based imaging.