Climate warming causes photobiont degradation and carbon starvation in a boreal climate sentinel lichen.

PREMISE: The long-term potential for acclimation by lichens to changing climates is poorly known, despite their prominent roles in forested ecosystems. Although often considered "extremophiles," lichens may not readily acclimate to novel climates well beyond historical norms. In a previous study (Smith et al., 2018), Evernia mesomorpha transplants in a whole-ecosystem climate change experiment showed drastic mass loss after 1 yr of warming and drying; however, the causes of this mass loss were not addressed. METHODS: We examined the causes of this warming-induced mass loss by measuring physiological, functional, and reproductive attributes of lichen transplants. RESULTS: Severe loss of mass and physiological function occurred above +2°C of experimental warming. Loss of algal symbionts ("bleaching") and turnover in algal community compositions increased with temperature and were the clearest impacts of experimental warming. Enhanced CO2 had no significant physiological or symbiont composition effects. The functional loss of algal photobionts led to significant loss of mass and specific thallus mass (STM), which in turn reduced water-holding capacity (WHC). Although algal genotypes remained detectable in thalli exposed to higher stress, within-thallus photobiont communities shifted in composition toward greater diversity. CONCLUSIONS: The strong negative impacts of warming and/or lower humidity on Evernia mesomorpha were driven by a loss of photobiont activity. Analogous to the effects of climate change on corals, the balance of symbiont carbon metabolism in lichens is central to their resilience to changing conditions.

Posttranslational regulation of transporters important for symbiotic interactions.

Coordinated sharing of nutritional resources is a central feature of symbiotic interactions, and, despite the importance of this topic, many questions remain concerning the identification, activity, and regulation of transporter proteins involved. Recent progress in obtaining genome and transcriptome sequences for symbiotic organisms provides a wealth of information on plant, fungal, and bacterial transporters that can be applied to these questions. In this update, we focus on legume-rhizobia and mycorrhizal symbioses and how transporters at the symbiotic interfaces can be regulated at the protein level. We point out areas where more research is needed and ways that an understanding of transporter mechanism and energetics can focus hypotheses. Protein phosphorylation is a predominant mechanism of posttranslational regulation of transporters in general and at the symbiotic interface specifically. Other mechanisms of transporter regulation, such as protein-protein interaction, including transporter multimerization, polar localization, and regulation by pH and membrane potential are also important at the symbiotic interface. Most of the transporters that function in the symbiotic interface are members of transporter families; we bring in relevant information on posttranslational regulation within transporter families to help generate hypotheses for transporter regulation at the symbiotic interface.

Analysis and modeling of Blastomyces dermatitidis environmental prevalence in Minnesota using soil collected to compare basal and outbreak levels.

Outbreaks of blastomycosis, caused by the fungus Blastomyces dermatitidis, occur in endemic areas of the United States and Canada but the geographic range of blastomycosis is expanding. Previous studies inferred the location of B. dermatitidis through epidemiologic data associated with outbreaks because culture of B. dermatitidis from the environment is often unsuccessful. In this study, we used a culture-independent, PCR-based method to identify B. dermatitidis DNA in environmental samples using the BAD1 promoter region. We tested 250 environmental samples collected in Minnesota, either associated with blastomycosis outbreaks or environmental samples collected from high- and low-endemic regions to determine basal prevalence of B. dermatitidis in the environment. We identified a fifth BAD1 promoter haplotype of B. dermatitidis prevalent in Minnesota. Ecological niche analysis identified latitude, longitude, elevation, and site classification as environmental parameters associated with the presence of B. dermatitidis Using this analysis, a Random Forest model predicted B. dermatitidis presence in basal environmental samples with 75% accuracy. These data support use of culture-independent, PCR-based environmental sampling to track spread into new regions and to characterize the unknown B. dermatitidis environmental niche.Importance Upon inhalation of spores from the fungus Blastomyces dermatitidis from the environment, humans and animals can develop the disease blastomycosis. Based on disease epidemiology, B. dermatitidis is known to be endemic in the United States and Canada around the Great Lakes and in the Ohio and Mississippi River Valleys but is starting to emerge in other areas. B. dermatitidis is extremely difficult to culture from the environment so little is known about the environmental reservoirs for this pathogen. We used a culture-independent PCR-based assay to identify the presence of B. dermatitidis DNA in soil samples from Minnesota. By combining molecular data with ecological niche modeling, we were able to predict the presence of B. dermatitidis in environmental samples with 75% accuracy and to define characteristics of the B. dermatitidis environmental niche. Importantly, we showed the effectiveness of using a PCR-based assay to identify B. dermatitidis in environmental samples.

Understanding transport processes in lichen, Azolla-cyanobacteria, ectomycorrhiza, endomycorrhiza, and rhizobia-legume symbiotic interactions.

Intimate interactions between photosynthetic and non-photosynthetic organisms require the orchestrated transfer of ions and metabolites between species. We review recent progress in identifying and characterizing the transport proteins involved in five mutualistic symbiotic interactions: lichens, Azolla-cyanobacteria, ectomycorrhiza, endomycorrhiza, and rhizobia-legumes. This review focuses on transporters for nitrogen and carbon and other solutes exchanged in the interactions. Their predicted functions are evaluated on the basis of their transport mechanism and prevailing transmembrane gradients of H + and transported substrates. The symbiotic interactions are presented in the assumed order from oldest to most recently evolved.

Identification of Pathogen Genomic Differences That Impact Human Immune Response and Disease during Cryptococcus neoformans Infection.

Patient outcomes during infection are due to a complex interplay between the quality of medical care, host immunity factors, and the infecting pathogen's characteristics. To probe the influence of pathogen genotype on human survival, immune response, and other parameters of disease, we examined Cryptococcus neoformans isolates collected during the Cryptococcal Optimal Antiretroviral Therapy (ART) Timing (COAT) Trial in Uganda. We measured human participants' survival, meningitis disease parameters, immunologic phenotypes, and pathogen in vitro growth characteristics. We compared those clinical data to whole-genome sequences from 38 C. neoformans isolates of the most frequently observed sequence type (ST), ST93, in our Ugandan participant population and to sequences from an additional 18 strains of 9 other sequence types representing the known genetic diversity within the Ugandan Cryptococcus clinical isolates. We focused our analyses on 652 polymorphisms that were variable among the ST93 genomes, were not in centromeres or extreme telomeres, and were predicted to have a fitness effect. Logistic regression and principal component analysis identified 40 candidate Cryptococcus genes and 3 hypothetical RNAs associated with human survival, immunologic response, or clinical parameters. We infected mice with 17 available KN99α gene deletion strains for these candidate genes and found that 35% (6/17) directly influenced murine survival. Four of the six gene deletions that impacted murine survival were novel. Such bedside-to-bench translational research identifies important candidate genes for future studies on virulence-associated traits in human Cryptococcus infections.IMPORTANCE Even with the best available care, mortality rates in cryptococcal meningitis range from 20% to 60%. Disease is often due to infection by the fungus Cryptococcus neoformans and involves a complex interaction between the human host and the fungal pathogen. Although previous studies have suggested genetic differences in the pathogen impact human disease, it has proven quite difficult to identify the specific C. neoformans genes that impact the outcome of the human infection. Here, we take advantage of a Ugandan patient cohort infected with closely related C. neoformans strains to examine the role of pathogen genetic variants on several human disease characteristics. Using a pathogen whole-genome sequencing approach, we showed that 40 C. neoformans genes are associated with human disease. Surprisingly, many of these genes are specific to Cryptococcus and have unknown functions. We also show deletion of some of these genes alters disease in a mouse model of infection, confirming their role in disease. These findings are particularly important because they are the first to identify C. neoformans genes associated with human cryptococcal meningitis and lay the foundation for future studies that may lead to new treatment strategies aimed at reducing patient mortality.

The lichen symbiosis re-viewed through the genomes of Cladonia grayi and its algal partner Asterochloris glomerata.

BACKGROUND: Lichens, encompassing 20,000 known species, are symbioses between specialized fungi (mycobionts), mostly ascomycetes, and unicellular green algae or cyanobacteria (photobionts). Here we describe the first parallel genomic analysis of the mycobiont Cladonia grayi and of its green algal photobiont Asterochloris glomerata. We focus on genes/predicted proteins of potential symbiotic significance, sought by surveying proteins differentially activated during early stages of mycobiont and photobiont interaction in coculture, expanded or contracted protein families, and proteins with differential rates of evolution. RESULTS: A) In coculture, the fungus upregulated small secreted proteins, membrane transport proteins, signal transduction components, extracellular hydrolases and, notably, a ribitol transporter and an ammonium transporter, and the alga activated DNA metabolism, signal transduction, and expression of flagellar components. B) Expanded fungal protein families include heterokaryon incompatibility proteins, polyketide synthases, and a unique set of G-protein α subunit paralogs. Expanded algal protein families include carbohydrate active enzymes and a specific subclass of cytoplasmic carbonic anhydrases. The alga also appears to have acquired by horizontal gene transfer from prokaryotes novel archaeal ATPases and Desiccation-Related Proteins. Expanded in both symbionts are signal transduction components, ankyrin domain proteins and transcription factors involved in chromatin remodeling and stress responses. The fungal transportome is contracted, as are algal nitrate assimilation genes. C) In the mycobiont, slow-evolving proteins were enriched for components involved in protein translation, translocation and sorting. CONCLUSIONS: The surveyed genes affect stress resistance, signaling, genome reprogramming, nutritional and structural interactions. The alga carries many genes likely transferred horizontally through viruses, yet we found no evidence of inter-symbiont gene transfer. The presence in the photobiont of meiosis-specific genes supports the notion that sexual reproduction occurs in Asterochloris while they are free-living, a phenomenon with implications for the adaptability of lichens and the persistent autonomy of the symbionts. The diversity of the genes affecting the symbiosis suggests that lichens evolved by accretion of many scattered regulatory and structural changes rather than through introduction of a few key innovations. This predicts that paths to lichenization were variable in different phyla, which is consistent with the emerging consensus that ascolichens could have had a few independent origins.

The Mouse Inhalation Model of Cryptococcus neoformans Infection Recapitulates Strain Virulence in Humans and Shows that Closely Related Strains Can Possess Differential Virulence.

Cryptococcal meningitis (CM) causes high rates of HIV-related mortality, yet the Cryptococcus factors influencing patient outcome are not well understood. Pathogen-specific traits, such as the strain genotype and degree of antigen shedding, are associated with the clinical outcome, but the underlying biology remains elusive. In this study, we examined factors determining disease outcome in HIV-infected cryptococcal meningitis patients infected with Cryptococcus neoformans strains with the same multilocus sequence type (MLST). Both patient mortality and survival were observed during infections with the same sequence type. Disease outcome was not associated with the patient CD4 count. Patient mortality was associated with higher cryptococcal antigen levels, the cerebrospinal fluid (CSF) fungal burden by quantitative culture, and low CSF fungal clearance. The virulence of a subset of clinical strains with the same sequence type was analyzed using a mouse inhalation model of cryptococcosis. We showed a strong association between human and mouse mortality rates, demonstrating that the mouse inhalation model recapitulates human infection. Similar to human infection, the ability to multiply in vivo, demonstrated by a high fungal burden in lung and brain tissues, was associated with mouse mortality. Mouse survival time was not associated with single C. neoformans virulence factors in vitro or in vivo; rather, a trend in survival time correlated with a suite of traits. These observations show that MLST-derived genotype similarities between C. neoformans strains do not necessarily translate into similar virulence either in the mouse model or in human patients. In addition, our results show that in vitro assays do not fully reproduce in vivo conditions that influence C. neoformans virulence.

MpAMT1;2 from Marchantia polymorpha is a High-Affinity, Plasma Membrane Ammonium Transporter.

Plant ammonium transporters in the AMT/MEP/Rh (ammonium transporter/methylammonium and ammonium permease/Rhesus factor) superfamily have only been previously characterized in flowering plants (angiosperms). Plant AMT1s are electrogenic, while plant AMT2s are electroneutral, and MEP and Rh transporters in other organisms are electroneutral. We analyzed the transport function of MpAMT1;2 from the basal land plant Marchantia polymorpha, a liverwort. MpAMT1;2 was shown to localize to the plasma membrane in Marchantia gametophyte thallus by stable transformation using a C-terminal citrine fusion. MpAMT1;2 expression was studied using quantitative real-time PCR and shown to be higher when plants were N deficient and lower when plants were grown on media containing ammonium, nitrate or the amino acid glutamine. Expression in Xenopus oocytes and analysis by electrophysiology revealed that MpAMT1;2 is an electrogenic ammonium transporter with a very high affinity for ammonium (7 µM at pH 5.6 and a membrane potential of -137 mV). A conserved inhibitory phosphorylation site identified in angiosperm AMT1s is also present in all AMT1s in Marchantia. Here we show that a phosphomimetic mutation T475D in MpAMT1;2 completely inhibits ammonium transport activity. The results indicate that MpAMT1;2 may be important for ammonium uptake into cells in the Marchantia thallus.

Evolution of Electrogenic Ammonium Transporters (AMTs).

The ammonium transporter gene family consists of three main clades, AMT, MEP, and Rh. The evolutionary history of the AMT/MEP/Rh gene family is characterized by multiple horizontal gene transfer events, gene family expansion and contraction, and gene loss; thus the gene tree for this family of transporters is unlike the organismal tree. The genomes of angiosperms contain genes for both electrogenic and electroneutral ammonium transporters, but it is not clear how far back in the land plant lineage electrogenic ammonium transporters occur. Here, we place Marchantia polymorpha ammonium transporters in the AMT/MEP/Rh phylogeny and we show that AMTs from the liverwort M. polymorpha are electrogenic. This information suggests that electrogenic ammonium transport evolved at least as early as the divergence of bryophytes in the land plant lineage.

Human Immune Response Varies by the Degree of Relative Cryptococcal Antigen Shedding.

Background. Cerebrospinal fluid (CSF) cryptococcal glucuronoxylomannan antigen (CrAg) titers generally correlate with quantitative fungal culture burden; however, correlation is not precise. Some patients have higher CrAg titers with lower fungal burdens and vice versa. We hypothesized that the relative discordancy between CrAg titer and quantitative culture burden reflects the relative degree of CrAg shedding by Cryptococcus neoformans and is associated with human immune responses. Methods. One hundred ninety human immunodeficiency virus-infected individuals with cryptococcal meningitis were enrolled in Uganda and South Africa. We compared initial CSF CrAg titers relative to their CSF quantitative cultures to determine low (n = 58), intermediate (n = 68), or high (n = 64) CrAg shedders. We compared cytokines measured by Luminex multiplex assay on cryopreserved CSF and 10-week mortality across shedding groups using linear and logistic regression and distribution of genotypes by multilocus sequence typing. Results. The relative degree of CrAg shedding was positively associated with increasing CSF levels of the following: interleukin (IL)-6, IL-7, IL-8, and tumor necrosis factor-α (each P < 0.01), which are all secreted by antigen-presenting cells and negatively associated with vascular endothelial growth factor (P = .01). In addition, IL-5, IL-13, granulocyte colony-stimulating factor, and macrophage chemotactic protein were decreased in low-CrAg shedders compared with intermediate shedders (each P ≤ .01). Type 1 T-helper cells (Th1) cytokine responses and 10-week mortality did not differ between the shedding groups. Cryptococcal genotypes were equally distributed across shedding groups. Conclusions. Discordancy between CrAg shedding and expected shedding based on quantitative fungal burden is associated with detectable immunologic differences in CSF, primarily among secreted cytokines and chemokines produced by antigen-presenting cells and Th2.

Increased Antifungal Drug Resistance in Clinical Isolates of Cryptococcus neoformans in Uganda.

Cryptococcal antigen screening is recommended among people living with AIDS when entering HIV care with a CD4 count of <100 cells/µl, and preemptive fluconazole monotherapy treatment is recommended for those with subclinical cryptococcal antigenemia. Yet, knowledge is limited of current antimicrobial resistance in Africa. We examined antifungal drug susceptibility in 198 clinical isolates collected from Kampala, Uganda, between 2010 and 2014 using the CLSI broth microdilution assay. In comparison with two previous studies from 1998 to 1999 that reported an MIC50 of 4 µg/ml and an MIC90 of 8 µg/ml prior to widespread human fluconazole and agricultural azole fungicide usage, we report an upward shift in the fluconazole MIC50 to 8 µg/ml and an MIC90 value of 32 µg/ml, with 31% of isolates with a fluconazole MIC of ≥ 16 µg/ml. We observed an amphotericin B MIC50 of 0.5 µg/ml and an MIC90 of 1 µg/ml, of which 99.5% of isolates (197 of 198 isolates) were still susceptible. No correlation between MIC and clinical outcome was observed in the context of amphotericin B and fluconazole combination induction therapy. We also analyzed Cryptococcus susceptibility to sertraline, with an MIC50 of 4 µg/ml, suggesting that sertraline is a promising oral, low-cost, available, novel medication and a possible alternative to fluconazole. Although the CLSI broth microdilution assay is ideal to standardize results, limit human bias, and increase assay capacity, such assays are often inaccessible in low-income countries. Thus, we also developed and validated an assay that could easily be implemented in a resource-limited setting, with similar susceptibility results (P = 0.52).

The Cryptococcus neoformans transcriptome at the site of human meningitis.

UNLABELLED: Cryptococcus neoformans is the leading cause of fungal meningitis worldwide. Previous studies have characterized the cryptococcal transcriptome under various stress conditions, but a comprehensive profile of the C. neoformans transcriptome in the human host has not been attempted. Here, we extracted RNA from yeast cells taken directly from the cerebrospinal fluid (CSF) of two AIDS patients with cryptococcal meningitis prior to antifungal therapy. The patients were infected with strains of C. neoformans var. grubii of molecular type VNI and VNII. Using RNA-seq, we compared the transcriptional profiles of these strains under three environmental conditions (in vivo CSF, ex vivo CSF, and yeast extract-peptone-dextrose [YPD]). Although we identified a number of differentially expressed genes, single nucleotide variants, and novel genes that were unique to each strain, the overall expression patterns of the two strains were similar under the same environmental conditions. Specifically, yeast cells obtained directly from each patient's CSF were more metabolically active than cells that were incubated ex vivo in CSF. Compared with growth in YPD, some genes were identified as significantly upregulated in both in vivo and ex vivo CSF, and they were associated with genes previously recognized for contributing to pathogenicity. For example, genes with known stress response functions, such as RIM101, ENA1, and CFO1, were regulated similarly in the two clinical strains. Conversely, many genes that were differentially regulated between the two strains appeared to be transporters. These findings establish a platform for further studies of how this yeast survives and produces disease. IMPORTANCE: Cryptococcus neoformans, an environmental, opportunistic yeast, is annually responsible for an estimated million cases of meningitis and over 600,000 deaths, mostly among HIV-infected patients in sub-Saharan Africa and Asia. Using RNA-seq, we analyzed the gene expression of two strains of C. neoformans obtained from the cerebrospinal fluid (CSF) of infected patients, thus creating a comprehensive snapshot of the yeasts' genetic responses within the human body. By comparing the gene expression of each clinical strain under three conditions (in vivo CSF, ex vivo CSF, and laboratory culture), we identified genes and pathways that were uniquely regulated by exposure to CSF and likely crucial for the survival of C. neoformans in the central nervous system. Further analyses revealed genetic diversity between the strains, providing evidence for cryptococcal evolution and strain specificity. This ability to characterize transcription in vivo enables the elucidation of specific genetic responses that promote disease production and progression.

High-throughput genome sequencing of lichenizing fungi to assess gene loss in the ammonium transporter/ammonia permease gene family.

BACKGROUND: Horizontal gene transfer has shaped the evolution of the ammonium transporter/ammonia permease gene family. Horizontal transfers of ammonium transporter/ammonia permease genes into the fungi include one transfer from archaea to the filamentous ascomycetes associated with the adaptive radiation of the leotiomyceta. The horizontally transferred gene has subsequently been lost in most of the group but has been selectively retained in lichenizing fungi. However, some groups of lichens appear to have secondarily lost the archaeal ammonium transporter. Definitive assessment of gene loss can only be made via whole genome sequencing. RESULTS: Ammonium transporter/ammonia permease gene sequences were recovered from the assembled genomes of eight lichenizing fungi in key clades including the Caliciales, the Peltigerales, the Ostropomycetidae, the Acarosporomycetidae, the Verrucariales, the Arthoniomycetidae and the Lichinales. The genes recovered were included in a refined phylogenetic analysis. The hypothesis that lichens symbiotic with a nitrogen-fixing cyanobacterium as a primary photobiont or lichens living in high nitrogen environments lose the plant-like ammonium transporters was upheld, but did not account for additional losses of ammonium transporters/ammonia permeases in the lichens from the Acarosporomycetidae, Chaetotheriomycetes and Arthoniomycetes. In addition, the four ammonium transporter/ammonia permease genes from Cladonia grayi were shown to be functional by expressing the lichen genes in a strain of Saccharomyces cerevisiae in which all three native ammonium transporters were deleted, and assaying for growth on limiting ammonia as a sole nitrogen source. CONCLUSIONS: Given sufficient coverage, next-generation sequencing technology can definitively address the loss of a gene in a genome when using environmental DNA isolated from lichen thalli collected from their natural habitats. Lichen-forming fungi have been losing ammonium transporters/ammonia permease genes at a slower rate than the most closely related non-lichenized lineages. These horizontally transferred genes in the Cladonia grayi genome encode functional ammonium transporters/ammonia permeases.

Cryptococcal genotype influences immunologic response and human clinical outcome after meningitis.

UNLABELLED: In sub-Saharan Africa, cryptococcal meningitis (CM) continues to be a predominant cause of AIDS-related mortality. Understanding virulence and improving clinical treatments remain important. To characterize the role of the fungal strain genotype in clinical disease, we analyzed 140 Cryptococcus isolates from 111 Ugandans with AIDS and CM. Isolates consisted of 107 nonredundant Cryptococcus neoformans var. grubii strains and 8 C. neoformans var. grubii/neoformans hybrid strains. Multilocus sequence typing (MLST) was used to characterize genotypes, yielding 15 sequence types and 4 clonal clusters. The largest clonal cluster consisted of 74 isolates. The results of Burst and phylogenetic analysis suggested that the C. neoformans var. grubii strains could be separated into three nonredundant evolutionary groups (Burst group 1 to group 3). Patient mortality was differentially associated with the different evolutionary groups (P = 0.04), with the highest mortality observed among Burst group 1, Burst group 2, and hybrid strains. Compared to Burst group 3 strains, Burst group 1 strains were associated with higher mortality (P = 0.02), exhibited increased capsule shedding (P = 0.02), and elicited a more pronounced Th(2) response during ex vivo cytokine release assays with strain-specific capsule stimulation (P = 0.02). The results of these analyses suggest that cryptococcal strain variation can be an important determinant of human immune responses and mortality. IMPORTANCE: Cryptococcus neoformans is a common life-threatening human fungal pathogen that is responsible for an estimated 1 million cases of meningitis in HIV-infected patients annually. Virulence factors that are important in human disease have been identified, yet the impacts of the fungal strain genotype on virulence and outcomes of human infection remain poorly understood. Using an analysis of strain variation based on in vitro assays and clinical data from Ugandans living with AIDS and cryptococcal infection, we report that strain genotype predicts the type of immune response and mortality risk. These studies suggest that knowledge of the strain genotype during human infections could be used to predict disease outcomes and lead to improved treatment approaches aimed at targeting the specific combination of pathogen virulence and host response.

Multiple horizontal gene transfers of ammonium transporters/ammonia permeases from prokaryotes to eukaryotes: toward a new functional and evolutionary classification.

The proteins of the ammonium transporter/methylammonium permease/Rhesus factor family (AMT/MEP/Rh family) are responsible for the movement of ammonia or ammonium ions across the cell membrane. Although it has been established that the Rh proteins are distantly related to the other members of the family, the evolutionary history of the AMT/MEP/Rh family remains unclear. Here, we use phylogenetic analysis to infer the evolutionary history of this family of proteins across 191 genomes representing all main lineages of life and to provide a new classification of the proteins in this family. Our phylogenetic analysis suggests that what has heretofore been conceived of as a protein family with two clades (AMT/MEP and Rh) is instead a protein family with three clades (AMT, MEP, and Rh). We show that the AMT/MEP/Rh family illustrates two contrasting modes of gene transmission: The AMT family as defined here exhibits vertical gene transfer (i.e., standard parent-to-offspring inheritance), whereas the MEP family as defined here is characterized by several ancient independent horizontal gene transfers (HGTs). These ancient HGT events include a gene replacement during the early evolution of the fungi, which could be a defining trait for the kingdom Fungi, a gene gain from hyperthermophilic chemoautolithotrophic prokaryotes during the early evolution of land plants (Embryophyta), and an independent gain of this same gene in the filamentous ascomycetes (Pezizomycotina) that was subsequently lost in most lineages but retained in even distantly related lichenized fungi. This recircumscription of the ammonium transporters/ammonia permeases family into MEP and AMT families informs the debate on the mechanism of transport in these proteins and on the nature of the transported molecule because published crystal structures of proteins from the MEP and Rh clades may not be representative of the AMT clade. The clades as depicted in this phylogenetic study appear to correspond to functionally different groups, with AMTs and ammonia permeases forming two distinct and possibly monophyletic groups.

RNA silencing of mycotoxin production in Aspergillus and Fusarium species.

Mycotoxins are natural fungal products that are defined by their harmful effects on humans and animals. Aflatoxin contamination of maize by Aspergillus species and trichothecene contamination of small grains by Fusarium species are two of the most severe mycotoxin problems in the United States. We are investigating RNA silencing in an effort to identify novel ways to control mycotoxin contamination of crops. Transformation of two Aspergilli (A. flavus and A. parasiticus) and a Fusarium (F. graminearum) with inverted repeat transgenes (IRT) containing sequences of mycotoxin-specific regulatory genes suppressed mycotoxin production in all three plant-pathogenic fungi. This atoxigenic phenotype was stable during infection on corn and wheat, and importantly, F. graminearum IRT strains were less virulent on wheat than were wild type. The IRT did not alter physiological characteristics of the fungi, such as spore production and growth rate on solid media. These results indicate that RNA silencing exists in Aspergillus and Fusarium plant pathogens and suggest that RNA silencing technology may be a useful tool for eliminating mycotoxin contamination of agricultural products.