The 1 ml conidial suspension (107 spores/ml) of the XS11 strain was added into 100 ml liquid CM and shaken for 3 days. The fresh hyphae were collected by filtration from the liquid shake cultures using a single-layer of Miracloth (Millipore) and were washed by sterile water three times. Then the vegetative hyphae were switched to liquid GMM with 10 mM of each of the sole nitrogen sources, including nitrate (NO3–) or ammonium (NH4+) at 25°C for 24 h. The fresh hyphae were collected by filtration from the liquid shake cultures using a single-layer of Miracloth (Millipore) and frozen in liquid nitrogen immediately. All of the fungal samples were ground to a fine powder using a mortar and pestle in liquid nitrogen. Total RNA was extracted by TRIzol reagent (Invitrogen) and purified with a PureLink RNA minikit (Ambion) in accordance with the manufacturers’ instructions.
Miracloth
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, Japan, Ireland
Miracloth is a high-quality filtration material made from pure cellulose. It is designed to provide efficient and reliable separation of solid and liquid components in a variety of laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Potato dextrose agar (pda), by BD (4 mentions)
Protease inhibitor cocktail, by Roche (4 mentions)
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Rneasy mini kit, by Qiagen (4 mentions)
Nd 1000 spectrophotometer, by Thermo Fisher Scientific (4 mentions)
Potato dextrose agar (pda), by Thermo Fisher Scientific (3 mentions)
Ab9050, by Abcam (3 mentions)
Potato dextrose broth (pdb), by BD (3 mentions)
Dneasy plant mini kit, by Qiagen (3 mentions)
Plant protease inhibitor cocktail, by Merck Group (3 mentions)
Driselase, by Merck Group (3 mentions)
Multidrop combi, by Thermo Fisher Scientific (3 mentions)
Pherastar fsx, by BMG Labtech (3 mentions)
Tween 20, by Merck Group (3 mentions)
Dneasy powerplant pro kit, by Qiagen (3 mentions)
Plant protease cocktail, by Merck Group (2 mentions)
α gfp, by Merck Group (2 mentions)
F1804, by Merck Group (2 mentions)
Glass coverslip, by Thermo Fisher Scientific (2 mentions)
Mini beadbeater, by Biospec (2 mentions)
380 protocols using miracloth
1
Nitrogen Source Regulation of Fungal Transcripts
2
Fungal Protein Extraction and Analysis
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For total fungal protein extraction, fungal mycelia were grown in PDB at 22°C, 120 rpm for 7 days and harvested by filtering through three layers of sterile Mira cloth (EMD Millipore Corp, Germany). Tissues were snap freezed in liquid nitrogen and stored in -80°C for future use. The fungal tissue was grounded to fine powder and resuspended in Tris-Glycine buffer pH 8.3 (3g Trizma, Sigma-Aldrich, USA and 14.4 g Glycine, Sigma-Aldrich, USA dissolved in 1 L MQ). Resuspension was then centrifuged at 16 x 1000g for 40 min at 4°C. The supernatant and cell lysate was collected separately. The total protein from the supernatant was separated on SDS-PAGE followed by immune blot with anti-Flag Ab (Cohesion biosciences, Singapore). For the extraction of secreted proteins, 7 days old grown mycelia in PDB was treated with 250 μM menadione (Sigma-Aldrich, USA), to mimic host induced oxidative stress condition. Axenic culture filtrate (CF) was collected and filtered through three layered Mira cloth (EMD Millipore Corp, Germany) to separate fungal mycelia. Further, CF was sequentially filtered with 0.45 and 0.22 μm Durapore PVDF membrane filters (Sigma-Aldrich, USA). Filtered CF was then concentrated using 3 kDa Amicon Ultra-15 Centrifugal Filter Units (Merck, USA). The concentrated secreted proteins were then separated on SDS-PAGE followed by immunoblot with anti-Flag Ab.
3
Fungal Transformation via Agrobacterium
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F. oxysporum was grown in minimal liquid medium containing 3% sucrose, 0.17% yeast nitrogen base and 100 mM KNO 3 in a shaker (175 rpm) at 25 °C for 5 days. Spores were filtered through two layers of miracloth (Merck; pore size of 22-25 lm), counted and a spore suspension was made with a final density of 2 Â 10 6 spores per milliliter. A. tumefaciens containing a binary vector for transformation to F. oxysporum, was grown to an OD of 0.45, then incubated for 6 h in Induction Medium (Mullins et al., 2001) (link) supplemented with 200 mM acetosyringone (AS) and then mixed in a 1:1 ratio with the fungal spore suspension. 100 ll of this mix was then pipetted on a 0.45-lm pore, 45-mm diameter nitrocellulose filter (Whatman) on either Potato Dextro Agar or CM supplemented with AS and grown for 3-5 days. Filters were then transferred to PDA plates containing 200 lg ml À1 cefotaxime.
After another 3-5 days of incubation spores were scraped off of the filters using liquid minimal medium and transferred to liquid minimal medium. After two to three days of incubation (175 rpm, 25 °C) spores were collected for sorting by filtering through two layers of miracloth (Merck; pore size of 22-25 lm).
After another 3-5 days of incubation spores were scraped off of the filters using liquid minimal medium and transferred to liquid minimal medium. After two to three days of incubation (175 rpm, 25 °C) spores were collected for sorting by filtering through two layers of miracloth (Merck; pore size of 22-25 lm).
4
Neurospora crassa Growth and Cultivation
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Neurospora crassa (strain name: ORS-SL6a, mating type: mat a, FGSC number: 4200) was used in this study and obtained from the Fungal Genetics Stock Center (FGSC, Manhattan, KS, USA). The fungus was maintained on Vogel’s Minimal (VM) agar. Fungal culture in glucose and Avicel media was performed as follows: a piece of N. crassa mycelia was inoculated onto VM agar media in a flask and cultured at 30 °C in darkness for 2 d and then at 25 °C in light for 12 d. Sterile deionized (DI) water (approximately 50 mL) was added into the flask and then shaken vigorously. Fungal suspension was filtered through two layers of Miracloth (EMD Millipore, Burlington, MA, USA) and then centrifuged at 3134× g for 5 min to pellet down spores. The spore pellet was resuspended in fresh sterile DI water. Spores (3 × 107) were inoculated into 30 mL VM liquid with 2% (w/v) glucose and incubated at 25 °C under constant light with shaking (200 rpm) for 24 h. Fungal mycelia were then collected by filtration through two layers of Miracloth (EMD Millipore) and washed with DI water. The washed mycelia were resuspended in 30 mL of fresh VM containing 2% (w/v) glucose or 2% (w/v) Avicel (Avicel PH-101, Sigma-Aldrich, St. Louis, MI, USA) and incubated at 25 °C under constant light with shaking (200 rpm) for 24, 48, and 72 h.
5
Co-immunoprecipitation of Plant Proteins
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In co-IP assays with proteins expressed in N. benthamiana Roq1 reconstitution assays, five 10 mm leaf discs were collected to form a sample. Total protein from the plant material ground to fine powder was extracted in 2 ml of the extraction buffer (10% glycerol, 100 mM Tris-HCl pH7.5, 5 mM MgCl2, 300 mM NaCl, 10 mM DTT, 0.5% NP-40, 2% PVPP, 1x Plant protease cocktail (11873580001, MilliporeSigma)). Lysates were centrifuged for 35 min at 4,500 x g and filtered through two layers of Miracloth (475855, MilliporeSigma). The 50 µl aliquots of the filtered supernatant were taken as input samples. Co-IP were conducted for 2 h with 12 μl a-HA affinity matrix (11815016001, MilliporeSigma) under constant rotation. Beads were collected by centrifugation at 4,000 x g for 1 min and washed four times in extraction buffer (without DTT and PVPP). All co-IP steps were conducted at 4°C. Beads and input samples were boiled at 95°C in 100 μl 2×Laemmli buffer for 10 min. Antibodies used for immunoblotting were α-GFP (11814460001, MilliporeSigma), α-HA (1:5000; c29f4, Cell Signalling), α-FLAG (1:5000; f1804, MilliporeSigma). Antibodies were used in dilution 1:5000 (TBST with 3% nonfat milk powder).
6
Isolation and Purification of Starch Granules
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Leaves of the wild type and mutants were separately collected and classified according to their age as young, mature (not shown), or old leaves when the whole plants were 4 weeks old. The leaf materials were frozen in liquid nitrogen and ground into a fine powder with a mortar and grinding mill. The powder was suspended in 40 mL extraction buffer (20 mM HEPES-KOH, pH 7.4, 0.4 mM EDTA, and 0.05% (v/v) Triton X-100) and filtered through Miracloth (475855-1R, Millipore Sigma, Darmstadt, Germany) before the filtrate was centrifuged at 4 °C, 1500× g for 5 min. Supernatants were discarded and the sediments were resuspended in 40 mL extraction buffer, which was filtered through 100 μm and 30 μm meshes before the filtrate was centrifuged as described above. The resulting sediments were again resuspended in 2 mL extraction buffer and slowly pipetted onto 5 mL Percoll (17-0891-01, GE Healthcare) and centrifuged at 4 °C, 1500× g for 15 min. The supernatants were discarded and the sediments/starch granules were washed with water three times and finally dried in a SpeedVac.
7
Genome Size Estimation by Flow Cytometry
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Genome size was estimated by flow cytometry using nuclei from red alder and tomato (Lycopersicon esculentum L.), a standard with a well-known genome size (Doležel et al. 1992 (link)). Samples from unexpanded red alder leaves kept at 4°C were chopped with a razor blade together with freshly harvested tomato leaves. Cold chopping buffer (15 mM HEPES, 1 mM EDTA, 80 mM KCl, 20 mM NaCl, 300 mM sucrose, 0.2% Triton X-100, 0.1% DTT, 0.5 mM spermine, and 0.25 mM polyvinylpyrolidone-40; modified from Dart et al. 2004 (link)) was added to the leaves prior to chopping, then again before filtering. Once filtered through 2 layers of Miracloth (Millipore Sigma) into a 1.5-mL microtube, the sample was centrifuged at 1,000 × g for 5 min. After removing the supernatant, the pellet was resuspended in 45 μL of 1.68 mM propidium iodide and 0.955 mL of a solution containing 100 mL MgSO4 buffer, 100 mg DTT, and 2.5 mL Triton X100. The MgSO4 buffer consisted of 0.25 g MgSO4·7 H2O, 0.37 g KCl, and 0.12 g of HEPES dissolved in 100 mL H2O with the pH adjusted to 8.0 (Arumuganathan and Earle 1991 (link)). The sample was then analyzed using a FACScan Flow Cytometer (BD Biosciences, San Jose, CA, USA). Peak positions corresponding to both sample and standard were recorded and the c-value for the red alder sample was computed.
8
Ultrastructural Analysis of Fungal Mycelia
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PAK1 mycelia from the single and dual cultures of S141+PAK1 were collected by filtering with miracloth (22–25 μm) (MilliporeSigma) after 24 and 48 h of cultivation. The mycelia collected were fixed with 2.5% glutaraldehyde (Electron Microscopy Sciences) in 0.1 M phosphate buffer (pH 7.2) at 4°C overnight, washed three times in phosphate buffer (4°C, pH 7.2), fixed in 1% osmium tetroxide (Electron Microscopy Sciences) for 2 h, washed three times in distilled water (room temperature), and then dehydrated in a graded acetone series (20, 40, 60, 80, and 100% three times). Specimens were dried in a critical point drying apparatus using liquid carbon dioxide as the medium. Hydrated specimens were affixed with conductive silver paint and coated with gold palladium (thickness ~15 nm). Samples were imaged with a field emission scanning electron microscope (FESEM_ZEISS model-Auriga, Coater _ Leica EM ACE600).
9
Isolation and Analysis of GFP-Tagged Proteins
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Co‐immunoprecipitation experiments were performed using the GFP‐Trap®_M kit (ChromoTek, Planegg, Germany). Proteins were extracted by grinding roots of 10‐d‐old Arabidopsis seedlings expressing pCAMV35S::GFP or pRXR1::262268845RXR1‐GFP in liquid N2, followed by homogenization of the powdered material in two volumes of ice‐cold buffer containing 25 mM HEPES‐KOH (pH 7.5), 1 mM EGTA, 1 mM EDTA, 20% (v/v) glycerol, 10 mM MgCl2, 25 mM NaF, 0.1% (v/v) Triton X‐100, 0.1% (w/v) polyvinylpolypyrrolidone, 1 mM phenylmethylsulfonyl fluoride (PMSF, MilliporeSigma), and 10 µg ml‐1 cOmplete™ EDTA‐free protease inhibitor cocktail (MilliporeSigma). Homogenates were centrifuged at 4°C and 16 000 g for 10 min, and the supernatant re‐centrifuged for 5 min and filtered through a layer of Miracloth (MilliporeSigma). Clarified extracts were incubated with pre‐washed GFP‐Trap®_M magnetic beads on an end‐to‐end tube rotator at 4°C for 1 h, and the beads subsequently washed according to manufacturer instructions. Bound protein was eluted with 200 mM glycine (pH 2.5) and immediately neutralized with 1 M Tris (pH 10.4). Composition of the eluates was analyzed by liquid chromatography–mass spectrometry (LC–MS) at the Charles W. Gehrke Proteomics Center (University of Missouri, Columbia, MO, USA).
10
Quantifying Fungal Spore Germination and Appressorium Formation
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Spores were harvested from 10-day-old cultures, filtered through two layers of Miracloth (Millipore Sigma), and resuspended to a concentration of 5 × 104 spores/ml in sterile water. For conidial germination, 20 μl of conidial suspension was placed on glass coverslips (Fisher Scientific, St. Louis, MO, USA) and incubated at 24°C in a humid chamber. The percentages of conidial germination and appressorium formation were assessed at 3, 5, 8, 12, and 20 h postincubation. Average mean values were determined from 150 conidia and performed in three independent experiments. Germ tube lengths were measured using ImageJ. All imaging was performed on a Zeiss LSM 800 confocal microscope, and images were converted using ImageJ (NIH).
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