The Functionality of the DC Pair in a Rhodopsin Guanylyl Cyclase from Catenaria anguillulae.

Rhodopsin guanylyl cyclases (RGCs) belong to the class of enzymerhodopsins catalyzing the transition from GTP into the second messenger cGMP, whereas light-regulation of enzyme activity is mediated by a membrane-bound microbial rhodopsin domain, that holds the catalytic center inactive in the dark. Structural determinants for activation of the rhodopsin moiety eventually leading to catalytic activity are largely unknown. Here, we investigate the mechanistic role of the D283-C259 (DC) pair that is hydrogen bonded via a water molecule as a crucial functional motif in the homodimeric C. anguillulae RGC. Based on a structural model of the DC pair in the retinal binding pocket obtained by MD simulation, we analyzed formation and kinetics of early and late photocycle intermediates of the rhodopsin domain wild type and specific DC pair mutants by combined UV-Vis and FTIR spectroscopy at ambient and cryo-temperatures. By assigning specific infrared bands to S-H vibrations of C259 we are able to show that the DC pair residues are tightly coupled. We show that deprotonation of D283 occurs already in the inactive L state as a prerequisite for M state formation, whereas structural changes of C259 occur in the active M state and early cryo-trapped intermediates. We propose a comprehensive molecular model for formation of the M state that activates the catalytic moiety. It involves light induced changes in bond strength and hydrogen bonding of the DC pair residues from the early J state to the active M state and explains the retarding effect of C259 mutants.

The inner mechanics of rhodopsin guanylyl cyclase during cGMP-formation revealed by real-time FTIR spectroscopy.

Enzymerhodopsins represent a recently discovered class of rhodopsins which includes histidine kinase rhodopsin, rhodopsin phosphodiesterases, and rhodopsin guanylyl cyclases (RGCs). The regulatory influence of the rhodopsin domain on the enzyme activity is only partially understood and holds the key for a deeper understanding of intra-molecular signaling pathways. Here, we present a UV-Vis and FTIR study about the light-induced dynamics of a RGC from the fungus Catenaria anguillulae, which provides insights into the catalytic process. After the spectroscopic characterization of the late rhodopsin photoproducts, we analyzed truncated variants and revealed the involvement of the cytosolic N-terminus in the structural rearrangements upon photo-activation of the protein. We tracked the catalytic reaction of RGC and the free GC domain independently by UV-light induced release of GTP from the photolabile NPE-GTP substrate. Our results show substrate binding to the dark-adapted RGC and GC alike and reveal differences between the constructs attributable to the regulatory influence of the rhodopsin on the conformation of the binding pocket. By monitoring the phosphate rearrangement during cGMP and pyrophosphate formation in light-activated RGC, we were able to confirm the M state as the active state of the protein. The described setup and experimental design enable real-time monitoring of substrate turnover in light-activated enzymes on a molecular scale, thus opening the pathway to a deeper understanding of enzyme activity and protein-protein interactions.

Phylogenomics of a new fungal phylum reveals multiple waves of reductive evolution across Holomycota.

Compared to multicellular fungi and unicellular yeasts, unicellular fungi with free-living flagellated stages (zoospores) remain poorly known and their phylogenetic position is often unresolved. Recently, rRNA gene phylogenetic analyses of two atypical parasitic fungi with amoeboid zoospores and long kinetosomes, the sanchytrids Amoeboradix gromovi and Sanchytrium tribonematis, showed that they formed a monophyletic group without close affinity with known fungal clades. Here, we sequence single-cell genomes for both species to assess their phylogenetic position and evolution. Phylogenomic analyses using different protein datasets and a comprehensive taxon sampling result in an almost fully-resolved fungal tree, with Chytridiomycota as sister to all other fungi, and sanchytrids forming a well-supported, fast-evolving clade sister to Blastocladiomycota. Comparative genomic analyses across fungi and their allies (Holomycota) reveal an atypically reduced metabolic repertoire for sanchytrids. We infer three main independent flagellum losses from the distribution of over 60 flagellum-specific proteins across Holomycota. Based on sanchytrids' phylogenetic position and unique traits, we propose the designation of a novel phylum, Sanchytriomycota. In addition, our results indicate that most of the hyphal morphogenesis gene repertoire of multicellular fungi had already evolved in early holomycotan lineages.

Solving a long-standing enigma: Myrmicinosporidium durum belongs to Blastocladiomycota, a phylum of primarily aquatic fungi.

Myrmicinosporidium durumHölldobler (1933) is a widely distributed fungal endoparasite of ants. However, little is known about its biology, ecology, or evolutionary history. Our study investigated the phylogenetics of this entomopathogenic fungus using a molecular approach. Samples of M. durum were obtained from infected Solenopsis fugax workers collected in Warsaw (Poland). Analyses of rDNA markers revealed that M. durum belongs to a phylum of primarily aquatic fungi, Blastocladiomycota. It is currently the only species from this group known to parasitise hymenopterans. Our findings have clarified this fungus' taxonomy and suggest future directions for research into its biology, ecology, and infection dynamics.

Genome-scale phylogenetic analyses confirm Olpidium as the closest living zoosporic fungus to the non-flagellated, terrestrial fungi.

The zoosporic obligate endoparasites, Olpidium, hold a pivotal position to the reconstruction of the flagellum loss in fungi, one of the key morphological transitions associated with the colonization of land by the early fungi. We generated genome and transcriptome data from non-axenic zoospores of Olpidium bornovanus and used a metagenome approach to extract phylogenetically informative fungal markers. Our phylogenetic reconstruction strongly supported Olpidium as the closest zoosporic relative of the non-flagellated terrestrial fungi. Super-alignment analyses resolved Olpidium as sister to the non-flagellated terrestrial fungi, whereas a super-tree approach recovered different placements of Olpidium, but without strong support. Further investigations detected little conflicting signal among the sampled markers but revealed a potential polytomy in early fungal evolution associated with the branching order among Olpidium, Zoopagomycota and Mucoromycota. The branches defining the evolutionary relationships of these lineages were characterized by short branch lengths and low phylogenetic content and received equivocal support for alternative phylogenetic hypotheses from individual markers. These nodes were marked by important morphological innovations, including the transition to hyphal growth and the loss of flagellum, which enabled early fungi to explore new niches and resulted in rapid and temporally concurrent Precambrian diversifications of the ancestors of several phyla of fungi.

The general architecture of black fly-parasite interactions: Parasitism in lotic systems at a continental scale.

We examined the general architecture of interactions between stream-dwelling larval black flies (Diptera: Simuliidae) and their common parasites in 1736 collections across North America. Mermithid nematodes (family Mermithidae), microsporidia (phylum Microsporidia), and the fungus Coelomycidium simulii Debaisieux (phylum Blastocladiomycota) infected larval black flies. We found similar continental distributions for these three parasite taxa across North America. At least one of these taxa was represented in 42.2% of all black fly collections. Species interactions in ecological networks typically imply that each link between species is equally important. By employing quantitative measures of host susceptibilities and parasite dependencies, we provide a more complete structure for host-parasite networks. The distribution of parasite dependencies and host susceptibilities were right-skewed, with low values indicating that most dependencies (parasites) and susceptibilities (hosts) were weak. Although regression analysis between host frequency and parasite incidence were highly significant, frequency analysis suggested that the distributions of parasites differ significantly among the four most common and closely related (same subgenus) species of hosts. A highly significant pattern of nestedness in our bipartite host-parasite network indicated that specialized parasites (i.e., those that interact with few host species) tend to occur as subsets of the most common hosts.

Molecular basis for GTP recognition by light-activated guanylate cyclase RhGC.

Cyclic guanosine 3',5'-monophosphate (cGMP) is an intracellular signalling molecule involved in many sensory and developmental processes. Synthesis of cGMP from GTP is catalysed by guanylate cyclase (GC) in a reaction analogous to cAMP formation by adenylate cyclase (AC). Although detailed structural information is available on the catalytic region of nucleotidyl cyclases (NCs) in various states, these atomic models do not provide a sufficient explanation for the substrate selectivity between GC and AC family members. Detailed structural information on the GC domain in its active conformation is largely missing, and no crystal structure of a GTP-bound wild-type GC domain has been published to date. Here, we describe the crystal structure of the catalytic domain of rhodopsin-GC (RhGC) from Catenaria anguillulae in complex with GTP at 1.7 Å resolution. Our study reveals the organization of a eukaryotic GC domain in its active conformation. We observe that the binding mode of the substrate GTP is similar to that of AC-ATP interaction, although surprisingly not all of the interactions predicted to be responsible for base recognition are present. The structure provides insights into potential mechanisms of substrate discrimination and activity regulation that may be common to all class III purine NCs. DATABASE: Structural data are available in Protein Data Bank database under the accession number 6SIR. ENZYMES: EC4.6.1.2.

Stimulation and Isolation of Paraphysoderma sedebokerense (Blastocladiomycota) Propagules and Their Infection Capacity Toward Their Host Under Different Physiological and Environmental Conditions.

Paraphysoderma sedebokerense (P. sedebokerense) (Blastocladiomycota) is a facultative pathogenic chytrid that causes irreversible damage to some green microalgae. Specific attacks leading to culture collapse under different conditions have only been described in the lucrative microalga Haematococcus pluvialis (H. pluvialis), while generating biomass for ketocarotenoid astaxanthin production, both indoors and outdoors. In order to manage the infection, parasite propagules (zoospores/amoeboid swarmers), the initiators of the disease, must be studied. Until now, no report on isolated P. sedebokerense propagules has been published. Here, we report on a reproducible method for the stimulation of P. sedebokerense propagule release and their isolation from fungal cultures in synthetic media and infected H. pluvialis cultures, and we further studied their development under different conditions. The isolated propagules featured different spore morphotypes, with coatless spherical spores and amoeboid swarmers being the most dominant in the first pulse of propagule release in both cultures. Inoculating the pure propagules with the host, in both the presence and absence of nitrogen, resulted in epidemic development in both green and red cells; however, in red cells, the epidemic developed more quickly in the presence of nitrogen. Biologically non-active autoclaved host cells were used to distinguish the initial stages of recognition from more progressive stages of the epidemics; on these cells, propagules encysted but did not develop further. These results prove the existence of heat-stable recognition sites on the host and an obligatory signal transduction from the host to support fungal cyst development. The propagule isolation method described herein is a breakthrough that will enable researchers to study the influence of different substances on the propagules, specifically as the initiators of the infection, and thus assist in the management of chytrid diseases. Moreover, it will be useful in studying host-parasite recognition and, therefore, will increase our understanding of the multiple chytrid infections found in nature.

Infection model for analyzing biological control of coffee rust using bacterial anti-fungal compounds.

Coffee rust is one of the main diseases that affect coffee plantations worldwide (Cressey, 2013 [10]). This causes an important economic impact in the coffee production industry in countries where coffee is an important part of the economy. A common method for combating this disease is using copper hydroxide as a fungicide, which can have damaging effects both on the coffee tree and on human health (Haddad et al., 2013 [13]). A novel method for biological control of coffee rust using bacteria has been proven to be an effective alternative to copper hydroxide fungicides as anti-fungal compounds (Haddad et al., 2009 [12]). In this paper, we develop and explore a spatial stochastic model for this interaction in a coffee plantation. We analyze equilibria for specific control strategies, as well as compute the basic reproductive number, R0, of individual coffee trees, conditions for local and global stability under specific conditions, parameter estimation of key parameters, as well as sensitivity analysis, and numerical experiments under local and global control strategies for key scenarios.

Rhodopsin-cyclases for photocontrol of cGMP/cAMP and 2.3 Å structure of the adenylyl cyclase domain.

The cyclic nucleotides cAMP and cGMP are important second messengers that orchestrate fundamental cellular responses. Here, we present the characterization of the rhodopsin-guanylyl cyclase from Catenaria anguillulae (CaRhGC), which produces cGMP in response to green light with a light to dark activity ratio >1000. After light excitation the putative signaling state forms with τ = 31 ms and decays with τ = 570 ms. Mutations (up to 6) within the nucleotide binding site generate rhodopsin-adenylyl cyclases (CaRhACs) of which the double mutated YFP-CaRhAC (E497K/C566D) is the most suitable for rapid cAMP production in neurons. Furthermore, the crystal structure of the ligand-bound AC domain (2.25 Å) reveals detailed information about the nucleotide binding mode within this recently discovered class of enzyme rhodopsin. Both YFP-CaRhGC and YFP-CaRhAC are favorable optogenetic tools for non-invasive, cell-selective, and spatio-temporally precise modulation of cAMP/cGMP with light.

New insights into the evolutionary history of Fungi from a 407 Ma Blastocladiomycota fossil showing a complex hyphal thallus.

Zoosporic fungi are key saprotrophs and parasites of plants, animals and other fungi, playing important roles in ecosystems. They comprise at least three phyla, of which two, Chytridiomycota and Blastocladiomycota, developed a range of thallus morphologies including branching hyphae. Here we describe Retesporangicus lyonii gen. et sp. nov., an exceptionally well preserved fossil, which is the earliest known to produce multiple sporangia on an expanded hyphal network. To better characterize the fungus we develop a new method to render surfaces from image stacks generated by confocal laser scanning microscopy. Here, the method helps to reveal thallus structure. Comparisons with cultures of living species and character state reconstructions analysed against recent molecular phylogenies of 24 modern zoosporic fungi indicate an affinity with Blastocladiomycota. We argue that in zoosporic fungi, kinds of filaments such as hyphae, rhizoids and rhizomycelium are developmentally similar structures adapted for varied functions including nutrient absorption and anchorage. The fossil is the earliest known type to develop hyphae which likely served as a saprotrophic adaptation to patchy resource availability. Evidence from the Rhynie chert provides our earliest insights into the biology of fungi and their roles in the environment. It demonstrates that zoosporic fungi were already diverse in 407 million-year-old terrestrial ecosystems.This article is part of a discussion meeting issue 'The Rhynie cherts: our earliest terrestrial ecosystem revisited'.

Expression, purification, and spectral tuning of RhoGC, a retinylidene/guanylyl cyclase fusion protein and optogenetics tool from the aquatic fungus Blastocladiella emersonii.

RhoGC is a rhodopsin (Rho)-guanylyl cyclase (GC) gene fusion molecule that is central to zoospore phototaxis in the aquatic fungus Blastocladiella emersonii It has generated considerable excitement because of its demonstrated potential as a tool for optogenetic manipulation of cell-signaling pathways involving cyclic nucleotides. However, a reliable method for expressing and purifying RhoGC is currently lacking. We present here an expression and purification system for isolation of the full-length RhoGC protein expressed in HEK293 cells in detergent solution. The protein exhibits robust light-dependent guanylyl cyclase activity, whereas a truncated form lacking the 17- to 20-kDa N-terminal domain is completely inactive under identical conditions. Moreover, we designed several RhoGC mutants to increase the utility of the protein for optogenetic studies. The first class we generated has altered absorption spectra designed for selective activation by different wavelengths of light. Two mutants were created with blue-shifted (E254D, λmax = 390 nm; D380N, λmax = 506 nm) and one with red-shifted (D380E, λmax = 533 nm) absorption maxima relative to the wild-type protein (λmax = 527 nm). We also engineered a double mutant, E497K/C566D, that changes the enzyme to a specific, light-stimulated adenylyl cyclase that catalyzes the formation of cAMP from ATP. We anticipate that this expression/purification system and these RhoGC mutants will facilitate mechanistic and structural exploration of this important enzyme.

Prevalence and dynamics of the zoosporic pathogen Catenaria uncinata in a natural population of the midge Glyptotendipes lobiferus.

A qPCR assay specific for zoospores of Catenaria uncinata, a fungal parasite in eggs of the midge Glyptotendipes lobiferus, was developed and used in parallel with traditional microscopic methods in a season-long study of a C. uncinata/G. lobiferus association in a local pond. Twenty-six consecutive weekly collections of egg masses were screened with a microscope to obtain percentages of infection and mortality in organogenetic egg masses and weekly water samples were processed by absolute quantification using qPCR to obtain estimates of zoospore density. Overall, 36.0% of G. lobiferus egg masses were infected to varying degrees and 11.2% of eggs were killed by C. uncinata. Continuous infection of egg masses occurred during a 6-wk period in May-June and a 7-wk period in September-October. Infection by C. uncinata was absent during a 10-week interval between periods of infection. Abrupt declines in zoospore density occurred during both infection periods and occurred only when water temperatures met or exceeded the viability threshold for zoospores (⩾31.0°C). The episodic death of zoospores during weeks in which egg infection and mortality levels were continuous likely resulted from distribution of zoospores throughout the water column and a temperature gradient in which zoospores sampled near the surface were subjected to lethal temperatures while non-sampled zoospores at lower depths were provided low temperature sanctuary. The hiatus of infection during the 10-week interval was likely due to lethal temperatures throughout the water column as average water temperatures exceeded 31.0°C over the period. A positive correlation between weekly zoospore densities obtained from qPCR and levels of infection/mortality in egg masses obtained from counts with a microscope supports the use of the qPCR assay alone in future studies that can rapidly and accurately determine parasite presence, prevalence and geographical range.

An ultrastructural study of Paraphysoderma sedebokerense (Blastocladiomycota), an epibiotic parasite of microalgae.

Successful algal cultivation for biofuel production is one path in the transition to a renewable energy economy. The green alga Scenedesmus dimorphus is a candidate for biofuel production, but is subject to parasitism and subsequent population crash when cultivated in open ponds. From an open pond cultivating S. dimorphus for biofuel production in New Mexico, USA, an amoeboid parasite was isolated, designated as isolate FD61, and its rDNA operon sequenced. A BLAST search for nuc 18S rDNA (18S) sequence similarity identified the parasite as Paraphysoderma sedebokerense (Blastocladiomycota). Here, we examine the ultrastructure of P. sedebokerense and compare it with that of a sister taxon, Physoderma maydis. The parasite has thin-walled vegetative sporangia and thick-walled resting sporangia. Our observations indicate that amoeboid swarmers are produced in the vegetative phase, while either amoeboid swarmers or zoospores are the product of meiosis in resting sporangia. Meiosis is confirmed by the presence of synaptonemal complexes in resting sporangia nuclei. Notably, P. sedebokerense has a Golgi apparatus with stacked cisternae, a feature reported for P. maydis, but which is absent in all other examined taxa in Blastocladiomycota. This report furthers our knowledge of the life cycle of P. sedebokerense.

An enigmatic fossil fungus from the 410 Ma Rhynie chert that resembles Macrochytrium (Chytridiomycota) and Blastocladiella (Blastocladiomycota).

Litter layers in the Lower Devonian (~ 410 Ma) Rhynie chert were inhabited by a wide variety of saprotrophic fungi, however, only a few of these organisms have been described formally. A new microfungus, Trewinomyces annulifer gen. et sp. nov., occurs as tufts on decaying land plant axes from the Rhynie chert. The fungus consists of an intramatrical rhizoidal system and an erect extramatrical hypha (stalk) that bears a single, terminal sporangium. One or two successive rings often are present in the stalk immediately below the sporangium base. Overall morphology of T. annulifer resembles the extant genera Macrochytrium (Chytridiomycota) and Blastocladiella (Blastocladiomycota). However, the rhizoids are septate or pseudoseptate, a feature not known in extant zoosporic fungi, and thus render the systematic affinities of T. annulifer unresolved. Trewinomyces annulifer offers a rare view of the morphology of a distinctive Early Devonian saprotrophic microfungus.

A Cyclic GMP-Dependent K+ Channel in the Blastocladiomycete Fungus Blastocladiella emersonii.

Phototaxis in flagellated zoospores of the aquatic fungus Blastocladiella emersonii depends on a novel photosensor, Blastocladiella emersonii GC1 (BeGC1), comprising a type I (microbial) rhodopsin fused to a guanylyl cyclase catalytic domain, that produces the conserved second messenger cyclic GMP (cGMP). The rapid and transient increase in cGMP levels during the exposure of zoospores to green light was shown to be necessary for phototaxis and dependent on both rhodopsin function and guanylyl cyclase activity. It is noteworthy that BeGC1 was localized to the zoospore eyespot apparatus, in agreement with its role in the phototactic response. A putative cyclic nucleotide-gated channel (BeCNG1) was also identified in the genome of the fungus and was implicated in flagellar beating via the action of a specific inhibitor (l-cis-diltiazem) that compromised zoospore motility. Here we show that B. emersonii expresses a K(+) channel that is activated by cGMP. The use of specific channel inhibitors confirmed the activation of the channel by cGMP and its K(+) selectivity. These characteristics are consistent with the function of an ion channel encoded by the BeCNG1 gene. Other blastocladiomycete fungi, such as Allomyces macrogynus and Catenaria anguillulae, possess genes encoding a similar K(+) channel and the rhodopsin-guanylyl cyclase fusion protein, while the genes encoding both these proteins are absent in nonflagellated fungi. The presence of these genes as a pair seems to be an exclusive feature of blastocladiomycete fungi. Taken together, these data demonstrate that the B. emersonii cGMP-activated K(+) channel is involved in the control of zoospore motility, most probably participating in the cGMP-signaling pathway for the phototactic response of the fungus.

Evidence for the involvement of surface carbohydrates in the recognition of Haematococcus pluvialis by the parasitic blastoclad Paraphysoderma sedebokerensis.

The unicellular green alga Haematococcus pluvialis (Chlorophyta, Volvocales) is currently the best commercial source of the natural red ketocarotenoid astaxanthin. Paraphysoderma sedebokerensis (Blastocladiomycota), a parasitic blastoclad that is specific for this microalga, was recently isolated and identified in our laboratory. In this study, we investigated the recognition process between the parasite and H. pluvialis. Obligatory requirements for recognition were identified as an ion concentration in the medium of 20 mM, the presence of calcium ions, and neutral to basic conditions; these requirements imply that a protein is involved in the process. In a search for potential lectin-sugar interactions as a major event in the recognition process, we screened for exposed glycosidic moieties on the cell wall of the alga and on the parasite zoospore surface. Competition experiments with the appropriate lectins and monosugars identified Ricinus communis agglutinin (RCA(120)) as the lectin that recognizes Gal-N-acetyl-d-glucosamine, an oligosaccharide located on the host. We propose that an RCA(120)-like lectin-sugar interaction mediates the highly specific interaction between the blastocladian parasite and its algal host.

[Yield loss model and yield loss mechanism of high-yielding summer maize infected by Physoderma maydis].

A total of 21 different disease-grading summer maize groups were formed by fixed-point natural infection of maize brown spot in the field, and mass loss estimation models of single ear mass and 100-grain mass were constructed by stepwise regression with DPS software. The mass loss estimation models of single ear and 100-grain were Y = -4.012 + 0.377X1 - 0.228X2 + 0.694X3 - 0.144X4 and Y = -4.536 + 0.173X1 + 0.188X2 + 0.248X3 - 0.034X4, respectively, where Y was yield loss rate, X1 was the disease index at flowering stage, X2 was the disease index at pollination stage, X3 was the disease index at filling stage, and X4 was the disease index at dough stage. The measured relationships between the disease indices at different growth stages and the mass loss for single ear and 100-grain coincided well with the modeling results. Maize brown spot directly affected the net photosynthetic rate of ear height leaf and the activities of RuBP carboxylase and PEP carboxylase. The higher the disease-grade, the lower the net photosynthetic rate and the activities of the two enzymes were.

Molecular phylogeny of the Blastocladiomycota (Fungi) based on nuclear ribosomal DNA.

The Blastocladiomycota is a recently described phylum of ecologically diverse zoosporic fungi whose species have not been thoroughly sampled and placed within a molecular phylogeny. In this study, we investigated the phylogeny of the Blastocladiomycota based on ribosomal DNA sequences from strains identified by traditional morphological and ultrastructural characters. Our results support the monophyly of the Coelomomycetaceae and Physodermataceae but the Blastocladiaceae and Catenariaceae are paraphyletic or polyphyletic. The data support two clades within Allomyces with strains identified as Allomyces arbusculus in both clades, suggesting that species concepts in Allomyces are in need of revision. A clade of Catenaria species isolated from midge larvae group separately from other Catenaria species, suggesting that this genus may need revision. In the Physodermataceae, Urophlyctis species cluster with a clade of Physoderma species. The algal parasite Paraphysoderma sedebokerensis nom. prov. clusters sister to other taxa in the Physodermataceae. Catenomyces persicinus, which has been classified in the Catenariaceae, groups with the Chytridiomycota rather than Blastocladiomycota. The rDNA operon seems to be suitable for classification within the Blastocladiomycota and distinguishes among genera; however, this region alone is not suitable to determine the position of the Blastocladiomycota among other basal fungal phyla with statistical support. A focused effort to find and isolate, or directly amplify DNA from additional taxa will be necessary to evaluate diversity in this phylum. We provide this rDNA phylogeny as a preliminary framework to guide further taxon and gene sampling and to facilitate future ecological, morphological, and systematic studies.