Te containing liquid AMM supplemented with increasing concentrations of DTT. Plates were incubated at 37uC for three days without shaking. The medium was then aspirated, and the hyphae adhering to the base of 1317923 the well were stained with 0.5 (w/v) methylene blue for one hour at 37uC. After removing the methylene blue solution, the adherent hyphae were rinsed with sterile water and dried prior to photographing. Sensitivity to tunicamycin (100 mg/ml) and brefeldin A (5?5 mg/ml) was determined by spotting conidia into each well of a 24-well plate containing AMM with increasing concentrations of the compound and incubating for 2? days at 37uC. For Title Loaded From File Analysis of hyphal growth, conidia were spot-plated onto the surface of a plate containing AMM agar and radial growth was Title Loaded From File monitored over a four-day incubation period at 37uC. The rate of radial growth was calculated as the colony diameter on day four minus the initial colony diameter after the first 24 hours of incubation divided by the incubation period.Analysis of Conidiophore DevelopmentFor analysis of conidiophore morphology, conidia were inoculated onto the edge of an OSM agar plug. A glass coverslip was placed on top of the plug and incubated for three days at 37uC. The coverslips were removed, mounted on a glass slide, and condiophores were observed using bright-field microscopy. For analysis of conidia morphology, wt and the DsrgA isolates were incubated on OSM plates for ten days at 37uC in tissue culture flasks; the flasks were 1315463 then removed and incubated at roomtemperature (RT) for seven days (RT incubation facilitated the conidiation of DsrgA isolate C). Conidia were then harvested from the plates and analyzed microscopically.Analysis of Intracellular Localization by GFP-TaggingPCR primers used to construct a GFP-srgA expression cassette are listed in Table S1. Total DNA was extracted from overnight cultures of wt A. fumigatus and srgA was PCR amplified using primers 824 and 825. The PCR product was then inserted into the NdeI and NotI sites of p538, a GFP-fusion cassette driven by the Aspergillus nidulans gpdA promoter [39], thus generating p626. Plasmid 626 was then ectopically introduced into the wt strain CBS144.89. The intracellular localization of the fusion protein was then determined by inoculating conidia from the GFP-SrgA A. fumigatus strain onto a glass coverslip submerged in liquid AMM and incubating overnight at 37uC. Coverslips, with adhered germlings on the surface, were then inverted and mounted on a glass slide. Images were acquired with a Zeiss LSM710 confocal with an Axio Observer Z1 set for GFP detection. Images of developing conidiophores were acquired using an Olympus IX71 inverted microscope set for GFP detection.G. mellonella Infection ModelG. mellonella larvae in the final instar stages were obtained from Vanderhorst, Inc (St. Marys, OH). Twelve larvae per group, weighing between 250?50 milligrams, were inoculated with conidia from either wt A. fumigatus or one of the DsrgA isolates. Five microliters of a 16108 conidia/ml saline suspension (56105 conidia) were injected into the last left pro-leg of each larva using a Hamilton syringe (Hamilton Company, Nevada). Six larvae were included in a control group, with each larva receiving an inoculum of five microliters of saline. The larvae were placed in petri dishessec4 Homolog in A. fumigatusand incubated in the dark at 37uC for five days. Larvae were examined daily and mortality was defined as lack of movement upon.Te containing liquid AMM supplemented with increasing concentrations of DTT. Plates were incubated at 37uC for three days without shaking. The medium was then aspirated, and the hyphae adhering to the base of 1317923 the well were stained with 0.5 (w/v) methylene blue for one hour at 37uC. After removing the methylene blue solution, the adherent hyphae were rinsed with sterile water and dried prior to photographing. Sensitivity to tunicamycin (100 mg/ml) and brefeldin A (5?5 mg/ml) was determined by spotting conidia into each well of a 24-well plate containing AMM with increasing concentrations of the compound and incubating for 2? days at 37uC. For analysis of hyphal growth, conidia were spot-plated onto the surface of a plate containing AMM agar and radial growth was monitored over a four-day incubation period at 37uC. The rate of radial growth was calculated as the colony diameter on day four minus the initial colony diameter after the first 24 hours of incubation divided by the incubation period.Analysis of Conidiophore DevelopmentFor analysis of conidiophore morphology, conidia were inoculated onto the edge of an OSM agar plug. A glass coverslip was placed on top of the plug and incubated for three days at 37uC. The coverslips were removed, mounted on a glass slide, and condiophores were observed using bright-field microscopy. For analysis of conidia morphology, wt and the DsrgA isolates were incubated on OSM plates for ten days at 37uC in tissue culture flasks; the flasks were 1315463 then removed and incubated at roomtemperature (RT) for seven days (RT incubation facilitated the conidiation of DsrgA isolate C). Conidia were then harvested from the plates and analyzed microscopically.Analysis of Intracellular Localization by GFP-TaggingPCR primers used to construct a GFP-srgA expression cassette are listed in Table S1. Total DNA was extracted from overnight cultures of wt A. fumigatus and srgA was PCR amplified using primers 824 and 825. The PCR product was then inserted into the NdeI and NotI sites of p538, a GFP-fusion cassette driven by the Aspergillus nidulans gpdA promoter [39], thus generating p626. Plasmid 626 was then ectopically introduced into the wt strain CBS144.89. The intracellular localization of the fusion protein was then determined by inoculating conidia from the GFP-SrgA A. fumigatus strain onto a glass coverslip submerged in liquid AMM and incubating overnight at 37uC. Coverslips, with adhered germlings on the surface, were then inverted and mounted on a glass slide. Images were acquired with a Zeiss LSM710 confocal with an Axio Observer Z1 set for GFP detection. Images of developing conidiophores were acquired using an Olympus IX71 inverted microscope set for GFP detection.G. mellonella Infection ModelG. mellonella larvae in the final instar stages were obtained from Vanderhorst, Inc (St. Marys, OH). Twelve larvae per group, weighing between 250?50 milligrams, were inoculated with conidia from either wt A. fumigatus or one of the DsrgA isolates. Five microliters of a 16108 conidia/ml saline suspension (56105 conidia) were injected into the last left pro-leg of each larva using a Hamilton syringe (Hamilton Company, Nevada). Six larvae were included in a control group, with each larva receiving an inoculum of five microliters of saline. The larvae were placed in petri dishessec4 Homolog in A. fumigatusand incubated in the dark at 37uC for five days. Larvae were examined daily and mortality was defined as lack of movement upon.
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