Hemocytometer

Cell viability was assessed using the Trypan blue assay. The cells were trypsinized using TrypLE™ Express Enzyme (Thermo Fisher Scientific, USA, 12,605,010) at 37 °C for 5 min. The cells were resuspended in 1 mL of PBS (Nacalai Tesque Inc., Japan, 27575-31) containing 1% FBS (Thermo Fisher Scientific, USA, 10270-106) to stop the enzyme reaction. One milliliter of 0.5% trypan blue dye solution (Nacalai Tesque Inc., Japan, 29853-34) was added, the cells were immediately loaded on a hemocytometer (Corning, USA, 480,200), and the number of viable cells per 1 × 1 mm square was counted under a stereo microscope.

The M. oryzae strain KV1 was used as the wild-type strain throughout this research (23 (link)) (see Table S1 in the supplemental material). This strain and its transformants were cultured on complete medium (CM; 1% [wt/vol] glucose, 0.2% [wt/vol] peptone, 0.1% [wt/vol] yeast extract, 0.1% [wt/vol] Casamino Acids, 0.1% trace elements, 0.1% vitamin solution, and 1× nitrate salts) and incubated at 25°C under 12-h light/dark cycles for 5 to 12 days. For DNA and RNA extraction, all strains were grown on CM liquid medium at 25°C with agitation for 48 h. For sporulation rates, strains were grown on at least three independent CM plates. After 12 days of growth, spores were harvested and counted using a hemocytometer (Corning). To observe vegetative mycelial growth under stress, 10 mM H₂O₂ and 150 μM menadione were individually added to CM agar medium. A mycelial disc (3.5 mm in diameter) of each strain was inoculated on stress medium containing either H₂O₂ or menadione, and the growth rate was assessed by measuring culture diameters after 5 days of growth (unless otherwise stated). Plate images were taken with an Epson perfection V700 photo scanner. For routine cloning, the Escherichia coli DH5α strain was grown in 2×YT broth at 37°C. The Saccharomyces cerevisiae yeast strain BY4741 was grown in synthetic dropout (SD) medium as described previously (31 (link)) at 30°C.

Guy11 was used as the wild type (WT) isolate for these studies [31] (link) and all mutant strains mentioned in this study were generated from the WT parental strain (Table S1). Standard procedures for the culture and storage of M. oryzae were used, as described in [47] (link). Strains were maintained on complete medium (CM), as described previously [38] (link). 85 mm diameter plates (unless otherwise stated) were incubated at 24°C under 12 hrs light/dark cycles. Plate images were taken with a Sony Cyber-shot digital camera, 14.1 mega pixels, after 10 days of growth (unless otherwise stated). For sporulation rates, strains were grown on at least three independent CM plates. After 12 days of growth, the spores were harvested and counted using a hemocytometer (Corning). Appressorial development assays were performed, as described previously [38] (link), on hydrophobic microscope coverslips (Fisherbrand). Average values were determined from 50 spores, performed in triplicate [38] (link).

GFs were seeded in 6-well (1 × 10⁵ cells/well) plates in growth medium with 10% FBS. Cells were left untreated or incubated with F. nucleatum at multiplicities of infection (MOIs) (F. nucleatum : cell of 10 : 1, 50 : 1, 100 : 1, 200 : 1, and 400 : 1). Cells were counted every other day using a hemocytometer (Corning, Corning, NY, USA) or an automated cell counter (Countstar, Shanghai, China). To assess the cell proliferation rate, cells were inoculated in 24-well plates (5 × 10⁴ cells/well) and treated with the indicated concentration of F. nucleatum. The 5-ethynyl-2′-deoxyuridine labeling assay was used to evaluate the cell proliferation rate according to the instructions of an EdU Apollo DNA in vitro kit (RiboBio, Guangzhou, China). The experiment was performed in sextuplicate and repeated three times.

Vegetative growth was assessed by measurement of colony diameter in plate cultures of different strains grown on different medium at 28°C for 10 days. The level of sporulation was assessed by harvesting conidia from the surface of 10-day-old strains grown on CM solid medium and determining the concentration of the resulting conidial suspension using a hemocytometer (Corning, China).
Appressorium formation of M. oryzae requires induction at 25°C in darkness for 24 or 48 h. For the rates of appressorium formation, conidial suspensions (5 × 10⁴ conidia ml⁻¹) were placed on hydrophobic coverslips for 24 h and measured by microscopic examination of at least 100 conidia. For inoculation to onion and barley epidermis surfaces in vitro, conidial suspensions (5 × 10⁴ conidia ml⁻¹) were carefully inoculated on onion or barley epidermis surfaces and incubated for 48 h. Then the appressorium were observed and photographed by optical microscope. Each test was repeated at least three times.

G. mellonella larvae were obtained through Vanderhorst Wholesale, St. Marys, Ohio, USA. C. neoformans strain H99 (serotype A) was kept frozen in 20% glycerol stocks and subcultured into Sabouraud dextrose broth for 48 hours at 30°C prior to each experiment. The yeast cells were washed twice with PBS, counted using a hemocytometer (Corning, New York, USA), and adjusted to 10⁶ cells/ml.
A. gambiae (Keele strain) mosquitoes were maintained on sugar solution at 27°C and 70% humidity with a 12-hour light to dark cycle according to standard rearing condition. P. falciparum NF54 (Walter Reed National Military Medical Center, Bethesda) infectious gametocyte cultures were provided by the Johns Hopkins Malaria Research Institute Parasite Core Facility and were diluted to 0.05% gametocytemia with naïve human blood before feeding to the mosquitoes using an artificial glass membrane feeder as established in [84 (link)].