Phase-specific transcriptional patterns of the oomycete pathogen Phytophthora sojae unravel genes essential for asexual development and pathogenic processes.

Oomycetes are filamentous microorganisms easily mistaken as fungi but vastly differ in physiology, biochemistry, and genetics. This commonly-held misconception lead to a reduced effectiveness by using conventional fungicides to control oomycetes, thus it demands the identification of novel functional genes as target for precisely design oomycetes-specific microbicide. The present study initially analyzed the available transcriptome data of the model oomycete pathogen, Phytophthora sojae, and constructed an expression matrix of 10,953 genes across the stages of asexual development and host infection. Hierarchical clustering, specificity, and diversity analyses revealed a more pronounced transcriptional plasticity during the stages of asexual development than that in host infection, which drew our attention by particularly focusing on transcripts in asexual development stage to eventually clustered them into 6 phase-specific expression modules. Three of which respectively possessing a serine/threonine phosphatase (PP2C) expressed during the mycelial and sporangium stages, a histidine kinase (HK) expressed during the zoospore and cyst stages, and a bZIP transcription factor (bZIP32) exclusive to the cyst germination stage were selected for down-stream functional validation. In this way, we demonstrated that PP2C, HK, and bZIP32 play significant roles in P. sojae asexual development and virulence. Thus, these findings provide a foundation for further gene functional annotation in oomycetes and crop disease management.

Leaf Mycobiome and Mycotoxin Profile of Warm-Season Grasses Structured by Plant Species, Geography, and Apparent Black-Stroma Fungal Structure.

Grasses harbor diverse fungi, including some that produce mycotoxins or other secondary metabolites. Recently, Florida cattle farmers reported cattle illness, while the cattle were grazing on warm-season grass pastures, that was not attributable to common causes, such as nutritional imbalances or nitrate toxicity. To understand correlations between grass mycobiome and mycotoxin production, we investigated the mycobiomes associated with five prominent, perennial forage and weed grasses [Paspalum notatum Flügge, Cynodon dactylon (L.) Pers., Paspalum nicorae Parodi, Sporobolus indicus (L.) R. Br., and Andropogon virginicus (L.)] collected from six Florida pastures actively grazed by livestock. Black fungal stromata of Myriogenospora and Balansia were observed on P. notatum and S. indicus leaves and were investigated. High-throughput amplicon sequencing was applied to delineate leaf mycobiomes. Mycotoxins from P. notatum leaves were inspected using liquid chromatography-mass spectrometry (LC-MS/MS). Grass species, cultivars, and geographic localities interactively affected fungal community assemblies of asymptomatic leaves. Among the grass species, the greatest fungal richness was detected in the weed S. indicus. The black fungal structures of P. notatum leaves were dominated by the genus Myriogenospora, while those of S. indicus were codominated by the genus Balansia and a hypermycoparasitic fungus of the genus Clonostachys. When comparing mycotoxins detected in P. notatum leaves with and without M. atramentosa, emodin, an anthraquinone, was the only compound which was significantly different (P < 0.05). Understanding the leaf mycobiome and the mycotoxins it may produce in warm-season grasses has important implications for how these associations lead to secondary metabolite production and their subsequent impact on animal health. IMPORTANCE The leaf mycobiome of forage grasses can have a major impact on their mycotoxin contents of forage and subsequently affect livestock health. Despite the importance of the cattle industry in warm-climate regions, such as Florida, studies have been primarily limited to temperate forage systems. Our study provides a holistic view of leaf fungi considering epibiotic, endophytic, and hypermycoparasitic associations with five perennial, warm-season forage and weed grasses. We highlight that plant identity and geographic location interactively affect leaf fungal community composition. Yeasts appeared to be an overlooked fungal group in healthy forage mycobiomes. Furthermore, we detected high emodin quantities in the leaves of a widely planted forage species (P. notatum) whenever epibiotic fungi occurred. Our study demonstrated the importance of identifying fungal communities, ecological roles, and secondary metabolites in perennial, warm-season grasses and their potential for interfering with livestock health.

Microecological insight to fungal structure and key fungal communities regulating nitrogen transformation based on spatial heterogeneity during cow manure composting by multi-angle and multi-aspect analyses.

Composting is the mainstream technology for the treatment of agricultural solid waste, but limited efforts were made to investigate fungal composition and its contributions to nitrogen transformation in different depths of compost. In this study, spatial distributions of fungi were analyzed using high throughput sequencing by multi-angle analyses, and the key fungal communities determining nitrogen transformation were quantified and identified by multi-aspect analyses during cow manure composting. Multi-angle analyses showed that fungal structure, biomarkers and trophic mode composition varied in different layers, revealing that spatial heterogeneity is the distinctive attribute of composting system. Ascomycota and Basidiomycota were dominant phyla during composting, the two phyla peaked in top and bottom layer respectively. At mesophilic stage, Tremellales, and unclassified Ascomycota (order) were biomarkers in top and middle layer respectively, and so were Remersonia, Pyrenochaetopsis, and Wallemia in bottom layer by LEfSe analysis. Based on multi-aspect analyses, Unclassified Dothideomycetes mainly affected NH4+-N transformation both in top (1.2816***) and middle layers (1.1726*). Trichocladium asperum (0.9536***) and Zopfiella (-0.9484***) mainly affected TN transformation in top layer. Guehomyces pullulans (-0.9684**) and Preussia (-1.0508**) regulated NO3--N transformation in middle layer. Thermomyces lanuginosus (0.7127***) and Typhula sp. UW973129 (0.7298***) were the key species promoting TN and C/N transformation in bottom layer, respectively. Interestingly, different fungal communities showed a complex network interaction driving nitrogen transformation, and the abundance of microbial community could be conducive to characterizing nitrogen transformation in the vertical space of composting.

Tetracycline hydrochloride-stressed succession in microbial communities during aerobic composting: Insights into bacterial and fungal structures.

Available information that whether antibiotics affect the succession in microbial communities during aerobic composting remains limited. Thus, this work investigated the dynamic changes in bacterial and fungal structures during aerobic composting amended with tetracycline hydrochloride (TCH: 0, 50, 150 and 300 mg kg-1). Composting phases significantly affected bacterial and fungal communities, but only fungi strongly responded to antibiotics, while bacteria did not. Firmicutes, Proteobacteria, Bacteroidota and Actinobacteriota were primary bacterial phylum. Neocallimastigomycota was dominant fungal phylum at temperature-heating phase, then Basidiomycota and Ascomycota became main fungal phylum at thermophilic and temperature-colling phases. Low TCH concentration promoted Chytridiomycota growth, while high TCH concentration inhibited mostly fungal activity in TCH-amended composting. Nitrogen species were critical factors controlling the succession in bacterial and fungal communities during composting process. These results cast a new light on understanding about microbial function during aerobic composting.

Diversity, Dispersal and Mode of Reproduction of Amanita exitialis in Southern China.

Amanita exitialis is a poisonous mushroom and has caused many deaths in southern China. In this study, we collected 118 fruiting bodies of A. exitialis from seven different sites in Guangdong Province in southern China and investigated their genetic relationships using 14 polymorphic molecular markers. These 14 markers grouped the 118 fruiting bodies into 20 multilocus genotypes. Among these 20 genotypes, eight were each found only once while the remaining 12 were each represented by two to 54 fruiting bodies. Interestingly, among the 12 shared genotypes, four were shared between/among local populations that were separated by as far as over 80 km, a result consistent with secondary homothallic reproduction and long-distance spore dispersal. Despite the observed gene flow, significant genetic differentiations were found among the local populations, primarily due to the over-representation of certain genotypes within individual local populations. STRUCTURE analyses revealed that the 118 fruiting bodies belonged to three genetic clusters, consistent with divergence within this species in this geographic region. Interestingly, we found an excess of heterozygous individuals at both the local and the total sample level, suggesting potential inbreeding depression and heterozygous advantage in these populations of A. exitialis. We discuss the implications of our results for understanding the life cycle, dispersal, and evolution of this poisonous mushroom.

Comparative Assessment of Antifungal and Antibacterial Activities of Crude Extracts of the King Tuber Culinary-Medicinal Mushroom, Pleurotus tuber-regium (Agaricomycetes) from Cameroon.

Antifungal and antibacterial activities of crude extracts of carpophore compared with those of sclerotium of Pleurotus tuber-regium were investigated on 11 species of bacterial and 3 fungal human pathogens. The minimal inhibitory concentration (MIC) of carpophore extract was recorded to be 12.5 mg/mL on Bacillus subtilis, Enterococcus faecalis, Staphylococcus epidermidis, Escherichia cloacae, Proteus mirabilis, P. vulgaris, Klebsiella oxytoca, and K. aerogenes and 6.25 mg/mL on Staphylococcus aureus, Escherichia coli, and Mycobacterium smegmatis as well as on all three species of fungal pathogens including Candida albicans, Aspergillus fumigatus, and A. ochraceus. In comparison, the MIC of sclerotium was recorded to be 12.5 mg/mL on Bacillus subtilis and Klebsiella aerogenes; 6.25 mg/mL on Enterococcus faecalis, Staphylococcus aureus, S. epidermidis, Enterobacter cloacae, E. coli, Mycobacterium smegmatis, Proteus mirabilis, P. vulgaris, and Klebsiella oxytoca; and 3.13 mg/mL on the three fungal pathogens. Based on the abovementioned figures, it appears that strains of pathogenic fungi tested are much more sensitive to crude extracts than the abovementioned bacteria. In fact, antimicrobial activities of crude extracts of P. tuber-regium, no matter whether it is that of the carpophore or its sclerotium, are in general stronger on human pathogenic fungi than bacteria. These figures also demonstrate that crude extracts of sclerotium show a higher antimicrobial activity than that of carpophore. Carpophores and sclerotia of P. tuber-regium could therefore constitute a source of new molecules potentially more efficient than synthetic products against some human pathogenic fungi and bacteria.

Recognition of Fungal Components by the Host Immune System.

By being the first point of contact of the fungus with the host, the cell wall plays an important role in the pathogenesis, having many molecules that participate as antigens that are recognized by immune cells, and also that help the fungus to establish infection. The main molecules reported to trigger an immune response are chitin, glucans, oligosaccharides, proteins, melanin, phospholipids, and others, being present in the principal pathogenic fungi with clinical importance worldwide, such as Histoplasma capsulatum, Paracoccidioides brasiliensis, Aspergillus fumigatus, Candida albicans, Cryptococcus neoformans, Blastomyces dermatitidis, and Sporothrix schenckii. Knowledge and understanding of how the immune system recognizes and responds to fungal antigens are relevant for the future research and development of new diagnostic tools and treatments for the control of mycosis caused by these fungi.

The entomopathogenic fungus Beauveria bassiana produces microsclerotia-like pellets mediated by oxidative stress and peroxisome biogenesis.

Several filamentous fungi are known to produce macroscopic pigmented hyphal aggregates named sclerotia. In recent years, some entomopathogenic fungi were reported to produce small sclerotia termed 'microsclerotia', becoming new potential propagules for biocontrol strategies. In this study, we described the production of microsclerotia-like pellets by the entomopathogenic fungus Beauveria bassiana. The carbon: nitrogen ratio equal to or higher than 12.5:1 amended with Fe2+ induced the germination of conidia, producing hyphal aggregate that formed sclerotial structures in submerged liquid cultures. These aggregates were able to tolerate desiccation as they germinated and subsequently produced viable conidia. Conidia derived from microsclerotial aggregates formulated with diatomaceous earth effectively kill Tribolium castaneum larvae. Optical and transmission microscopical imaging, qPCR and spectrophotometric analysis revealed that an oxidative stress scenario is involved in conidial differentiation into microsclerotia-like pellets, inducing fungal antioxidant response with high peroxidase activity - mainly detected in peroxisomes and mitochondria - and progress with active peroxisome proliferation. The results provide clues about B. bassiana microsclerotial differentiation and indicate that these pigmented aggregates are promising propagules for production, formulation and potentially application in the control of soil-inhabiting arthropod pests.

Anti-Candidaalbicans germ tube antibodies reduce in vitro growth and biofilm formation of C. albicans / Los anticuerpos dirigidos contra los tubos germinales de Candida albicans reducen el crecimiento in vitro y la formación de biopelículas de C. albicans

Background: Invasive candidiasis by Candida albicans is associated with high morbidity and mortality, due in part to the late implementation of an appropriate antifungal therapy hindered by the lack of an early diagnosis. Aims: We aimed to evaluate the in vitro antifungal activity of the antibodies against C. albicans germ tubes (CAGTA) raised in a rabbit model of candidemia. Methods: We measured the effect of CAGTA activity by colorimetric XTT and crystal violet assays, and colony forming units count, both on C. albicans planktonic cells and during the course of biofilm formation and maturation. Viability and cell morphology were assessed by optical, fluorescent or scanning electron microscopy. Results: CAGTA ≥ 50 μg/ml caused a strong inhibition of C. albicans blastospores growth, and DiBAC fluorescent staining evidenced a fungicidal activity. Moreover, electron microscopy images revealed that CAGTA induced morphological alterations of the surface of C. albicans germ tubes grown free as well as in biofilm. Interestingly, CAGTA ≥ 80 μg/ml reduced the amount of C. albicans biofilm, and this effect started at the initial adhesion stage of the biofilm formation, during the first 90 min. Conclusions: This is the first report showing that CAGTA reduce C. albicans growth, and impair its metabolic activity and ability to form biofilm in vitro. The antigens recognized by CAGTA could be the basis for the development of immunization protocols that might protect against Candida infections

Antecedentes: La infección invasora por Candida albicans está asociada a altas tasas de morbimortalidad, en parte debido al retraso en la instauración de una terapia antifúngica adecuada, dificultada a su vez por la falta de un diagnóstico precoz. Objetivos: Evaluar la actividad antifúngica de los anticuerpos contra tubos germinales de C. albicans (CAGTA) obtenidos a partir de un modelo animal de candidemia en conejo. Métodos. El efecto de los CAGTA se evaluó mediante los ensayos colorimétricos XTT y cristal violeta, así como mediante el recuento de unidades formadoras de colonias, tanto en células planctónicas de C. albicans como en distintos estadios de formación y maduración de biopelículas. La viabilidad y la morfología de las células tratadas con CAGTA se determinó mediante microscopía óptica, de fluorescencia o electrónica (SEM). Resultados: Concentraciones de CAGTA ≥ 50 μg/ml generaban una fuerte inhibición del crecimiento de C. albicans, y su actividad se mostró fungicida. Los CAGTA producían alteraciones en la superficie de los tubos germinales desarrollados tanto a partir de células en suspensión como de células en biopelículas. Además, concentraciones de CAGTA ≥ 80 μg/ml redujeron la biomasa de biopelículas de Candida, y este efecto se desencadenaba en los primeros 90min de su formación. Conclusiones: Este es el primer estudio que demuestra la capacidad de los CAGTA para reducir el crecimiento de C. albicans y su actividad metabólica, así como para alterar la formación de biopelículas in vitro. Los antígenos reconocidos por los CAGTA podrían servir de base para el desarrollo de protocolos de inmunización protectores frente a infecciones por Candida

Anti-Candidaalbicans germ tube antibodies reduce in vitro growth and biofilm formation of C. albicans.

BACKGROUND: Invasive candidiasis by Candida albicans is associated with high morbidity and mortality, due in part to the late implementation of an appropriate antifungal therapy hindered by the lack of an early diagnosis. AIMS: We aimed to evaluate the in vitro antifungal activity of the antibodies against C. albicans germ tubes (CAGTA) raised in a rabbit model of candidemia. METHODS: We measured the effect of CAGTA activity by colorimetric XTT and crystal violet assays, and colony forming units count, both on C. albicans planktonic cells and during the course of biofilm formation and maturation. Viability and cell morphology were assessed by optical, fluorescent or scanning electron microscopy. RESULTS: CAGTA ≥50µg/ml caused a strong inhibition of C. albicans blastospores growth, and DiBAC fluorescent staining evidenced a fungicidal activity. Moreover, electron microscopy images revealed that CAGTA induced morphological alterations of the surface of C. albicans germ tubes grown free as well as in biofilm. Interestingly, CAGTA ≥80µg/ml reduced the amount of C. albicans biofilm, and this effect started at the initial adhesion stage of the biofilm formation, during the first 90min. CONCLUSIONS: This is the first report showing that CAGTA reduce C. albicans growth, and impair its metabolic activity and ability to form biofilm in vitro. The antigens recognized by CAGTA could be the basis for the development of immunization protocols that might protect against Candida infections.

Cryopreparation and Electron Tomography of Yeast Cells.

Three-dimensional imaging of cells using electron tomography enables analysis of cell structure at unprecedented resolution. The preparation of cells for tomography using rapid freezing followed by freeze-substitution is an essential first step to ensure the optimal preservation of the cell structure for 3D studies. This protocol outlines a method for obtaining well-preserved cells using high-pressure freezing followed by freeze-substitution. We have found that this method is particularly well suited for electron tomography studies and has the added bonus of preserving antigenicity for immuno-electron microscopy. The steps involved in imaging cells and performing tomographic analysis of cellular structures are also outlined.

Building Cell Structures in Three Dimensions: Electron Tomography Methods for Budding Yeast.

Saccharomyces cerevisiae has been an important model system for numerous cellular, genetic, and molecular studies. However, this small eukaryote presents a challenge for imaging at the electron microscope level. Preparation of yeast using high-pressure freezing followed by freeze-substitution (HPF/FS) results in excellent preservation of cell structure in these difficult-to-fix samples. In particular, cells prepared by HPF/FS can be used for 3D electron tomography (ET) studies where optimum cell preservation is critical. Here, we discuss the advantages of using HPF/FS for ET and show examples of the utility of this method for building yeast cell structures in three dimensions.

Putative methyltransferase LaeA and transcription factor CreA are necessary for proper asexual development and controlling secondary metabolic gene cluster expression.

The morphological development of fungi is a complex process and is often coupled with secondary metabolite production. In this study, we assessed the function of putative methyltransferase LaeA and transcription factor CreA in controlling asexual development and secondary metabolic gene cluster expression in Penicillium oxalicum. The deletion of laeA (ΔlaeA) impaired the conidiation in P. oxalicum, with a downregulated expression of brlA. Overexpression of P. oxalicum brlA in ΔlaeA could upregulate brlA and abaA remarkably, but could not rescue the conidiation defect; therefore, brlA and abaA expression were necessary but not sufficient for conidiation. Deletion of creA in ΔlaeA background (ΔlaeAΔcreA) blocked conidiation with a white fluffy phenotype. Nutrient-rich medium could not rescue developmental defects in ΔlaeAΔcreA mutant but could rescue defects in ΔlaeA. Expression of 10 genes, namely, albA/wA, abrB/yA, arpA, aygA, arpA-like, arpB, arpB-like, rodA, rodA-like, and rodB, for pigmentation and spore wall protein genes was silenced in ΔlaeAΔcreA, whereas only six of them were downregulated in ΔlaeA. Among the 28 secondary metabolism gene clusters in P. oxalicum, four secondary metabolism gene clusters were silenced in ΔlaeA and two were also silenced in ΔbrlA mutant. A total of 10 physically linked and coregulated genes were distributed over five chromosomes in ΔlaeA. Six of these genes were located in subtelomeric regions, thus demonstrating a positional bias for LaeA-regulated clusters toward subtelomeric regions. All of silenced clusters located in subtelomeric regions were derepressed in ΔlaeAΔcreA, hence showing that lack of CreA could remediate the repression of gene clusters in ΔlaeA background. Results show that both putative methyltransferase LaeA and transcription factor CreA are necessary for proper asexual development and controlling secondary metabolic gene cluster expression.

Chromium immobilization by extra- and intraradical fungal structures of arbuscular mycorrhizal symbioses.

Arbuscular mycorrhizal (AM) fungi can enhance plant Cr tolerance through immobilizing Cr in mycorrhizal roots. However, the detailed processes and mechanisms are unclear. The present study focused on cellular distribution and speciation of Cr in both extraradical mycelium (ERM) and mycorrhizal roots exposed to Cr(VI) by using field emission scanning electron microscopy equipped with energy dispersive X-ray spectrometer (FE-SEM-EDS), scanning transmission soft X-ray microscopy (STXM) and X-ray absorption fine structure (XAFS) spectroscopy techniques. We found that amounts of particles (possibly extracellular polymeric substances, EPS) were produced on the AM fungal surface upon Cr(VI) stress, which contributed greatly to Cr(VI) reduction and immobilization. With EDS of the surface of AM fungi exposed to various Cr(VI) levels, a positive correlation between Cr and P was revealed, suggesting that phosphate groups might act as counter ions of Cr(III), which was also confirmed by the XAFS analysis. Besides, STXM and XAFS analyses showed that Cr(VI) was reduced to Cr(III) in AM fungal structures (arbuscules, intraradical mycelium, etc.) and cell walls in mycorrhizal roots, and complexed possibly with carboxyl groups or histidine analogues. The present work provided evidence of Cr immobilization on fungal surface and in fungal structures in mycorrhizal roots at a cellular level, and thus unraveled the underlying mechanisms by which AM symbiosis immobilize Cr.

Localization of the bioluminescence system in the pileus of Mycena chlorophos.

Mycena chlorophos, which is primarily distributed in Southeast Asia, is a luminous fungus that emits a bright green light from its pileus for about 2 days at approximately 20°C and high relative humidity. The distribution of bioluminescent tissues in the whole pileus and its sections was heterogeneous. The light intensity in the cap and the upper region of the gill was greater than that in the lower region of the gill. At the microscopic level, the light was predominantly emitted from the membranes of hymenium and basidia cells on the gill. The emission was both cell and region specific. The luminescence system was localized in the cell membrane, and a part of the system was on the cell membrane surface.

Ciclo de vida de corynelia tropica, patógeno foliar en podocarpus saligna / Life cycle of corynelia tropica, leaf pathogen on podocarpus saligna

El presente estudio tuvo como objetivo determinar el ciclo de vida de Corynelia tropica, patógeno de hojas y ramillas en mañío de hoja larga (Podocarpus saligna). Mensualmente se recolectaron ramillas de P. saligna. En la medida que se fueron detectando estadíos interesantes dentro del ciclo de vida de este patógeno, la frecuencia de las colectas se intensificó en forma quincenal, e incluso semanal. Se pudo constatar que C. tropica presentó un ciclo de vida anual muy definido, con gran regularidad en la aparición y desarrollo de sus estructuras fructíferas. Invariablemente el ciclo de vida se inició en la primera quincena de noviembre con la inoculación de los nuevos brotes de P. saligna. A inicios de enero se manifestaron los primeros síntomas de la infección y a comienzos de marzo ya eran notorias las estructuras del anamorfo. A fines de mayo las estructuras del teleomorfo se hacían protuberantes y se iniciaba la formación de los ascos. A fines de julio los ascocarpos presentaban forma y tamaño ya adulto, y las ascosporas al interior de los ascos se encontraban en proceso de maduración. A fines de octubre las ascosporas ya estaban maduras y su liberación a comienzos de noviembre, sincronizaba con la emergencia de los brotes de P. saligna, con lo que se daba inicio una vez más a un nuevo ciclo de vida de C. tropica. Se pudo constatar que además del follaje y ramas, este patógeno también atacaba a los frutos de P.saligna.

This study aimed to determine the life cycle of Corynelia tropica, pathogen leaves and twins in longleaf mañío (Podocarpus saligna). Monthly P. saligna twigs were collected. As they were detecting interesting stages in the life cycle of this pathogen, the frequency of collections were intensified fortnightly or even weekly. It was found that C. tropica presented a very defined, with great regularity in the occurrence and development of their fruiting structures annual life cycle. Invariably the life cycle began in the first half of November with the inoculation of new outbreaks of P. saligna. In early January the first symptoms of infections manifested and early March were already notorious the anamorph structure. In late May teleomorph structures became prominent and the formation of the asci began. In late July the ascocarps presented adult size and shape, and the ascospores within the asci were maturing. In late October ascospores were ripe and in early November, release synchronized with outbreaks of P. saligna, which was given start again a new life cycle of C. tropica. It was found that besides the foliage and branches, this pathogen also attacked the fruits of P. saligna.

Dominant negative RPW8.2 fusion proteins reveal the importance of haustorium-oriented protein trafficking for resistance against powdery mildew in Arabidopsis.

Powdery mildew fungi form feeding structures called haustoria inside epidermal cells of host plants to extract photosynthates for their epiphytic growth and reproduction. The haustorium is encased by an interfacial membrane termed the extrahaustorial membrane (EHM). The atypical resistance protein RPW8.2 from Arabidopsis is specifically targeted to the EHM where RPW8.2 activates haustorium-targeted (thus broad-spectrum) resistance against powdery mildew fungi. EHM-specific localization of RPW8.2 suggests the existence of an EHM-oriented protein/membrane trafficking pathway during EHM biogenesis. However, the importance of this specific trafficking pathway for host defense has not been evaluated via a genetic approach without affecting other trafficking pathways. Here, we report that expression of EHM-oriented, nonfunctional RPW8.2 chimeric proteins exerts dominant negative effect over functional RPW8.2 and potentially over other EHM-localized defense proteins, thereby compromising both RPW8.2-mediated and basal resistance to powdery mildew. Thus, our results highlight the importance of the EHM-oriented protein/membrane trafficking pathway for host resistance against haustorium-forming pathogens such as powdery mildew fungi.

GATA-Dependent Glutaminolysis Drives Appressorium Formation in Magnaporthe oryzae by Suppressing TOR Inhibition of cAMP/PKA Signaling.

Fungal plant pathogens are persistent and global food security threats. To invade their hosts they often form highly specialized infection structures, known as appressoria. The cAMP/ PKA- and MAP kinase-signaling cascades have been functionally delineated as positive-acting pathways required for appressorium development. Negative-acting regulatory pathways that block appressorial development are not known. Here, we present the first detailed evidence that the conserved Target of Rapamycin (TOR) signaling pathway is a powerful inhibitor of appressorium formation by the rice blast fungus Magnaporthe oryzae. We determined TOR signaling was activated in an M. oryzae mutant strain lacking a functional copy of the GATA transcription factor-encoding gene ASD4. Δasd4 mutant strains could not form appressoria and expressed GLN1, a glutamine synthetase-encoding orthologue silenced in wild type. Inappropriate expression of GLN1 increased the intracellular steady-state levels of glutamine in Δasd4 mutant strains during axenic growth when compared to wild type. Deleting GLN1 lowered glutamine levels and promoted appressorium formation by Δasd4 strains. Furthermore, glutamine is an agonist of TOR. Treating Δasd4 mutant strains with the specific TOR kinase inhibitor rapamycin restored appressorium development. Rapamycin was also shown to induce appressorium formation by wild type and Δcpka mutant strains on non-inductive hydrophilic surfaces but had no effect on the MAP kinase mutant Δpmk1. When taken together, we implicate Asd4 in regulating intracellular glutamine levels in order to modulate TOR inhibition of appressorium formation downstream of cPKA. This study thus provides novel insight into the metabolic mechanisms that underpin the highly regulated process of appressorium development.

Biological Activities of the Polysaccharides Produced from Different Sources of Xylaria nigripes (Ascomycetes), a Chinese Medicinal Fungus.

Xylaria nigripes, a local rare medicinal fungus, has multi-antioxidant activities owing to its water extraction as shown by previous research. However, the main indicator causing the antioxidant effect was not clear, so this research focused on the antioxidant activities from different sources of X. nigripes such as fruiting body polysaccharides, mycelium intracellular polysaccharides, mycelium extracellular polysaccharides, and their deproteinization products. The mycelium intracellular polysaccharide (XnIPS-1) from X. nigripes showed the highest reducing power of antioxidant activity, since it revealed the lowest IC50 values in all the assayed methodologies. The IC50 values of chelating ferrous ion ability, ABTS radical scavenging activity, and DPPH free radical scavenging were 1412, 174.25, and 351.56 µg/mL, respectively. In addition to these results, this research also explored the mechanism between polysaccharides and antioxidants compared by FT-IR analysis. The spectrum shows that the X. nigripes polysaccharide structure changed after the proteins were removed.