Regulation of polarised growth in fungi

Polarised growth in fungi occurs through the delivery of secretory vesicles along tracks formed by cytoskeletal elements to specific sites on the cell surface where they dock with a multiprotein structure called the exocyst before fusing with the plasmamembrane. The budding yeast, Saccharomyces cerevisiae has provided a useful model to investigate the mechanisms involved and their control. Cortical markers, provided by bud site selection pathways during budding, the septin ring during cytokinesis or the stimulation of the pheromone response receptors during mating, act through upstream signalling pathways to localise Cdc24, the GEF for the rho family GTPase, Cdc42. Cdc42 in its GTP-bound activates a multiprotein protein complex called the polarisome which nucleates actin cables along which the secretory vesicles are transported to the cell surface. Hyphae can elongate at a rate orders of magnitude faster than the extension of a yeast bud, so understanding hyphal growth will require substantial modification of the yeast paradigm. The rapid rate of hyphal growth is driven by a structure called the Spitzenkörper, located just behind the growing tip and which is rich in secretory vesicles. It is thought that secretory vesicles are delivered to the apical region where they accumulate in the Spitzenkörper. The Spitzenkörper then acts as vesicle supply centre in which vesicles exit the Spitzenkörper in all directions, but because of its proximity, the tip receives a greater concentration of vesicles per unit area than subapical regions. There are no obvious equivalents to the bud site selection pathway to provide a spatial landmark for polarised growth in hyphae. However, an emerging model is the way that the site of polarised growth in the fission yeast, Schizosaccharomyces pombe, is marked by delivery of the kelch repeat protein, Tea1, along microtubules. The relationship of the Spitzenkörper to the polarisome and the mechanisms that promote its formation are key questions that form the focus of current research

Fungal Traits Important for Soil Aggregation

Soil structure, the complex arrangement of soil into aggregates and pore spaces, is a key feature of soils and soil biota. Among them, filamentous saprobic fungi have well-documented effects on soil aggregation. However, it is unclear what properties, or traits, determine the overall positive effect of fungi on soil aggregation. To achieve progress, it would be helpful to systematically investigate a broad suite of fungal species for their trait expression and the relation of these traits to soil aggregation. Here, we apply a trait-based approach to a set of 15 traits measured under standardized conditions on 31 fungal strains including Ascomycota, Basidiomycota, and Mucoromycota, all isolated from the same soil. We find large differences among these fungi in their ability to aggregate soil, including neutral to positive effects, and we document large differences in trait expression among strains. We identify biomass density, i.e., the density with which a mycelium grows (positive effects), leucine aminopeptidase activity (negative effects) and phylogeny as important factors explaining differences in soil aggregate formation (SAF) among fungal strains; importantly, growth rate was not among the important traits. Our results point to a typical suite of traits characterizing fungi that are good soil aggregators, and our findings illustrate the power of employing a trait-based approach to unravel biological mechanisms underpinning soil aggregation. Such an approach could now be extended also to other soil biota groups. In an applied context of restoration and agriculture, such trait information can inform management, for example to prioritize practices that favor the expression of more desirable fungal traits

Can biochar ameliorate phosphorus deficiency and aluminium phytotoxicity in acid soils? : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy of Applied Science in Soil Science at Massey University, Palmerston North, New Zealand

The use of biochar as soil amendment to enhance soil functionality is being increasingly investigated, with particular attention given to its effects on the sustainable increase of crop production and carbon (C) sequestration. To date, however, limited research has attempted to unravel the effect of biochar on either the chemical and/or biological mobilization of the residual fraction of phosphorus (P) in soil. This fraction tends to accumulate as a result of long–term P fertilization in soils rich in aluminium (Al) and iron (Fe) oxy–hydroxides and short–range ordered aluminosilicates (i.e. allophane). There is also scant information on (i) how the speciation of soluble Al changes when biochar is applied to acid soils, and (ii) whether this application alleviates Al toxicity on plant roots. My objective in this study is therefore to determine the effect of different biochars with contrasting fertilizer and liming values on the chemistry, biology and nutrient fertility of acid mineral soils. Before studying the effect of different biochars on soil properties, several methodologies for measuring the liming properties and available nitrogen (N) in biochar were evaluated and modified where needed. For this, 19 biochars produced by pyrolysing a wide range of feedstocks under various production temperatures were used. Different pH–buffering capacity (pH–BC) methodologies – originally developed for soils (single vs multiple acid additions, short vs long equilibration times) – were tested, along with the methodology used to measure the liming equivalence. The methodologies were then validated by incubating over 10 d two acid soils (an Haplic Cambisol and an Andic Umbrisol) to which separated amendments of the 19 biochars were made at the rates estimated using both methodologies to target a final pH of 6.5 The results indicated that the relationship established between the pH–BC of the 19 biochars under study after 30–min equilibration (pH–BC30min) with a single addition of acid and that obtained after a 5–d equilibration (pH–BC5d) (predicted pH–BC5d = 2.2 × pH–BC30min + 20.4) allowed an adequate estimate of the liming potential of biochars. Similar results were found with the liming equivalent, and both methods were considered suitable to make the recommendation of the application rate. Acid hydrolysis using 6 M HCl has been proved adequate to determine available N in biochar. For this, hydrolysates of biochar are oxidized using potassium peroxodisulfate with a dilution factor of 600 so that chloride interferences are overcome and nitrate–N is then measured. This methodology, originally developed using biochars rich in N, proved not suitable for biochars with low N concentrations. Results obtained in this study have shown that a smaller dilution factor (242) is sufficiently adequate to overcome the chloride interferences while avoiding over–diluting the sample. In this study, we hypothesized that biochar can increase P availability to plant by stimulating the growth of arbuscular mycorrhizal fungi (AMF) hyphae. Therefore, methodologies to (i) estimate the length of fungal hyphae in soil and (ii) evaluate the transfer of P by AMF hyphae needed to be tested and modified where necessary. In this part of the study, three different biochars and two soil types were used. Two biochars were produced from chipped pine (Pinus radiata D. Don) branches at 450oC and 550°C (referred to as BP450 and BP550, respectively); and a third one from chipped weeping willow (Salix matsudana L.) branches at 550°C (referred to as BW550). The soils were two sil–andic Andosols of contrasting P status (Olsen P of 4.3 vs 33.3 mg kg–1, referred to as LP and HP soil, respectively). The traditional visual gridline intersection (VGI) method commonly used to measure the length of AMF hyphae distribution in soil was modified by (i) using a digital photomicrography technique (referred to as “digital gridline intersection” (DGI) method), and (ii) processing the images using ImageJ software (referred to as the “photomicrography–ImageJ processing” (PIP) method). These methods were first tested with known lengths of possum fur and then applied to measuring the hyphal length in the LP and HP soils after a 32 wk experiment growing Lotus pedunculatus cv barsille. The study confirmed that the use of digital photomicrography in conjunction with either the grid–line intersection principle or image processing (with ImageJ software) is a suitable method for the measurement of AMF hyphal lengths in soils. In addition, the traditional root study container that divides the plant growth medium into two sections – (i) a root zone to which both root and AMF hyphae have access and (ii) an hyphal zone to which only AMF hyphae have access – by a layer of nylon mesh was further modified by including a 3–mm thickness of tephra under the nylon mesh between two sections. This layer of tephra was found to be adequate to halt P diffusion from the HP soil to the LP soil for a plant growth period of 32 wk. Under such circumstances, the increase in P uptake by plant growth in a combination of a root zone of LP soil and a hyphal zone of HP soil compared with that in which both root and hyphal zones were filled with LP soil was only ascribed to the transfer of P from HP soil to LP soil by AMF hyphae. This novel root study container allows the biochar to be added to either the root zone or the hyphal zone and separates the effect of biochar on AMF hyphae development and P uptake from that on P content and availability (i.e., biochar rich in P; changes in soil pH). This device can contribute to discern whether biochar can influence AMF development and enhance P bioavailability. In order to investigate the feasibility of adding biochar to soils with high residual P so that this can become bioavailable, Lotus pedunculatus cv barsille was grown in LP and HP soils separately amended with BP450, BP550 and BW550 biochars at an application rate of 10 t ha–1 using the novel root study container for 32 wk without any further P and N fertilization. We found that (i) none of the tested biochars conferred any specific advantage to the HP soil; (ii) the addition of BW550 biochar to the LP soil increased plant growth by 59% and P uptake by 73%, while the pine–based biochar (e.g., BP450 and BP550) provided no extra nutrient uptake and no plant growth increase. This was ascribed to supplemental nutrients (especially P) from the BW550 biochar along with its liming effect and associated increase in P availability; (iii) biochar produced from BP450 biochar caused a 70% P uptake increase (and 40% plant growth increase) by stimulating AMF growth and accessing a high–P area (HP soil) to which the plant root had no access. More research is needed to discern the underlying mechanism. The liming effects of BW550 and BP550 biochars were further compared with those of lime chemicals (e.g., Ca(OH)2 and NaOH) in a short–term (10–d) incubation using two soils with contrasting pH–BC (an Haplic Cambisol and an Andic Umbrisol) to which these amendments were added. The two soils were first amended with BW550, BP550, Ca(OH)2 or NaOH at specific rates so that pH values of 5.4, 5.6, 5.8 and 6.4 were targeted and incubated at room temperature (25 oC) for 10 d. At the end of the incubation, a radical elongation bioassay using alfalfa (Medicago sativa L.) was carried out. Thereafter, soils were characterized with special attention to the Al chemistry, i.e. aqueous reactive Al fractionation and inorganic monomeric Al speciation. The final objective was to reveal the mechanisms through which these biochars alleviate Al toxicity on roots. Results showed that, for a specific soil, a smaller amount of BW550 biochar was required to increase the same unit of pH and reduce a similar amount of exchangeable Al compared with the amount required of BP550 biochar. The addition of BW550 biochar (at application rates < 9.1 %) and Ca(OH)2 stimulated alfalfa (Medicago sativa L.) seedling growth, whereas that of BP550 (at application rates > 2.4 %) and NaOH caused inhibition. The distinct responses of the root growth to the presence of Ca(OH)2 and BW550 biochar and to that of NaOH and BP550 biochar were explained by (i) a decrease in both inorganic monomeric Al (mainly in AlF2+ and Al3+) and colloidal Al, and (ii) an increase in aqueous Ca2+, in the former, as expected. In the latter there was (i) an increase in aqueous colloidal Al and Na+, and (ii) a decrease in soluble Ca2+. Thus, BW550 biochar was shown to be a more effective liming agent than was BP550 biochar. The information obtained in this thesis supports the use of biochar to manage high P affinity Andosols and acid soils, which are abundant in New Zealand. The technology of producing biochar from willow woodchips or feedstock alike with resultant solid products of high nutrient status and liming potential may contribute to the recycle of nutrients while increasing soil pH. Pine woodchips produced at relatively low temperature (e.g., 450oC) have been shown to enhance AMF abundance and functionality. Thus, biochar with specific environmental and agricultural purposes should be tailored accordingly. The root study container with a layer of “P diffusion break” and the measurement of AMF hyphal length using the photomicrography in conjunction with image software analysis (e.g., ImageJ) will advance studies of the responses of AMF to soil additives (e.g., biochar or green waste) and their associated enhancement of soil functions

Rapid turnover of hyphae of mycorrhizal fungi determined by AMS microanalysis of C-14

Processes in the soil remain among the least well-characterized components of the carbon cycle. Arbuscular mycorrhizal (AM) fungi are ubiquitous root symbionts in many terrestrial ecosystems and account for a large fraction of photosynthate in a wide range of ecosystems; they therefore play a key role in the terrestrial carbon cycle. A large part of the fungal mycelium is outside the root ( the extraradical mycelium, ERM) and, because of the dispersed growth pattern and the small diameter of the hyphae (<5 micrometers), exceptionally difficult to study quantitatively. Critically, the longevity of these. ne hyphae has never been measured, although it is assumed to be short. To quantify carbon turnover in these hyphae, we exposed mycorrhizal plants to fossil ("carbon-14 - dead") carbon dioxide and collected samples of ERM hyphae ( up to 116 micrograms) over the following 29 days. Analyses of their carbon-14 content by accelerator mass spectrometry (AMS) showed that most ERM hyphae of AM fungi live, on average, 5 to 6 days. This high turnover rate reveals a large and rapid mycorrhizal pathway of carbon in the soil carbon cycle

Functional Profiling of Transcription Factor Genes in Neurospora crassa.

Regulation of gene expression by DNA-binding transcription factors is essential for proper control of growth and development in all organisms. In this study, we annotate and characterize growth and developmental phenotypes for transcription factor genes in the model filamentous fungus Neurospora crassa We identified 312 transcription factor genes, corresponding to 3.2% of the protein coding genes in the genome. The largest class was the fungal-specific Zn2Cys6 (C6) binuclear cluster, with 135 members, followed by the highly conserved C2H2 zinc finger group, with 61 genes. Viable knockout mutants were produced for 273 genes, and complete growth and developmental phenotypic data are available for 242 strains, with 64% possessing at least one defect. The most prominent defect observed was in growth of basal hyphae (43% of mutants analyzed), followed by asexual sporulation (38%), and the various stages of sexual development (19%). Two growth or developmental defects were observed for 21% of the mutants, while 8% were defective in all three major phenotypes tested. Analysis of available mRNA expression data for a time course of sexual development revealed mutants with sexual phenotypes that correlate with transcription factor transcript abundance in wild type. Inspection of this data also implicated cryptic roles in sexual development for several cotranscribed transcription factor genes that do not produce a phenotype when mutated

Novel insights into host-fungal pathogen interactions derived from live-cell imaging

Acknowledgments The authors acknowledge funding from the Wellcome Trust (080088, 086827, 075470 and 099215) including a Wellcome Trust Strategic Award for Medical Mycology and Fungal Immunology 097377 and FP7-2007–2013 grant agreement HEALTH-F2-2010-260338–ALLFUN to NARG.Peer reviewedPublisher PD

Pythium species from rice roots differ in virulence, host colonization and nutritional profile

Background: Progressive yield decline in Philippine aerobic rice fields has been recently associated with three closely related Pythium spp., P. arrhenomanes, P. graminicola and P. inflatum. To understand their differential virulence towards rice seedlings, we conducted a comparative survey in which three isolates each of P. arrhenomanes, P. graminicola and P. inflatum were selected to investigate host colonization, host responses and carbon utilization profiles using histopathological analyses, phenoarrays, DNA quantifications and gene expression studies. Results: The isolate of the most virulent species, P. arrhenomanes, quickly colonized the outer and inner root tissues of rice seedlings, including the xylem, by which it possibly blocked the water transport and induced severe stunting, wilting and seedling death. The lower virulence of the tested P. graminicola and P. inflatum isolates seemed to be reflected in slower colonization processes, limited invasion of the vascular stele and less systemic spread, in which cell wall fortification appeared to play a role. Progressive hyphal invasions triggered the production of reactive oxygen species (ROS) and phenolic compounds, which was the strongest for the P. arrhenomanes isolate and was delayed or much weaker upon inoculation with the P. inflatum isolate. The necrosis marker OsJamyb seemed faster and stronger induced by the most virulent isolates. Although the isolate of P. inflatum was nutritionally the most versatile, the most virulent Pythium isolate appeared physiologically more adapted to its host, evidenced by its broad amino acid utilization profile, including D-amino acids, L-threonine and hydroxyl-L-proline. The latter two compounds have been implicated in plant defense and their use by P. arrhenomanes could therefore represent a part of its virulence strategy. Conclusions: This study illustrates that the differential virulence of rice-pathogenic P. arrhenomanes, P. graminicola and P. inflatum isolates is related to their root colonization capacity, the intensity of induced root responses and their ability to utilize amino acids in their colonization niche. Accordingly, this paper presents important knowledge concerning rice root infections by oomycetes, which could be helpful to further disentangle virulence tactics of soil-borne pathogens

The importance of subclasses of chitin synthase enzymes with myosin-like domains for the fitness of fungi

Acknowledgements TG and CF are funded by FEDER funds through the Operational Programme Competitiveness Factors – COMPETE and national funds by FCT – Foundation for Science and Technology under the strategic project UID/NEU/04539/2013. C.F. is a recipient of a postdoctoral fellowship from FCT-Fundação para a Ciência e Tecnologia (SFRH/BPD/63733/2009). NG is funded by The Wellcome Trust (080088, 086827, 075470, 099215 & 097377), the FungiBrain Marie Curie Network and the Medical Research Council (UK).Peer reviewe

TPLATE recruitment reveals endocytic dynamics at sites of symbiotic interface assembly in arbuscular mycorrhizal interactions

Introduction: Arbuscular mycorrhizal (AM) symbiosis between soil fungi and the majority of plants is based on a mutualistic exchange of organic and inorganic nutrients. This takes place inside root cortical cells that harbor an arbuscule: a highly branched intracellular fungal hypha enveloped by an extension of the host cell membrane—the perifungal membrane—which outlines a specialized symbiotic interface compartment. The perifungal membrane develops around each intracellular hypha as the symbiotic fungus proceeds across the root tissues; its biogenesis is the result of an extensive exocytic process and shows a few similarities with cell plate insertion which occurs at the end of somatic cytokinesis. Materials and Methods: We here analyzed the subcellular localization of a GFP fusion with TPLATE, a subunit of the endocytic TPLATE complex (TPC), a central actor in plant clathrin-mediated endocytosis with a role in cell plate anchoring with the parental plasma membrane. Results: Our observations demonstrate that Daucus carota and Medicago truncatula root organ cultures expressing a 35S::AtTPLATE-GFP construct accumulate strong fluorescent green signal at sites of symbiotic interface construction, along recently formed perifungal membranes and at sites of cell-to-cell hyphal passage between adjacent cortical cells, where the perifungal membrane fuses with the plasmalemma. Discussion: Our results strongly suggest that TPC-mediated endocytic processes are active during perifungal membrane interface biogenesis—alongside exocytic transport. This novel conclusion, which might be correlated to the accumulation of late endosomes in the vicinity of the developing interface, hints at the involvement of TPC-dependent membrane remodeling during the intracellular accommodation of AM fungi