N parisii ribosomal rna

N. parisii spores were prepared as previously described (57 (link)). A mixed population of DRH-1(2CARD) animals and non-transgenic siblings was synchronized at the L1 stage and plated on 6-cm NGM plates along with a mixture of OP50–1, M9, and one million spores for 30 h at 25°C. A minimum of four replicates (four plates) were set up per treatment. Three independent experiments were performed. To assess the presence of N. parisii meronts, animals were washed in M9 and fixed in 4% paraformaldehyde for 15 min prior to an overnight incubation at 47 °C with a FAM-conjugated FISH probe that hybridizes to N. parisii ribosomal RNA (Biosearch Technologies). Samples were sorted based on red fluorescence using the COPAS Biosort instrument (Union Biometrica) to obtain homogenous populations of either DRH-1(2CARD) animals or non-transgenic siblings. Green fluorescence was measured for each population using the COPAS Biosort instrument (Union Biometrica). For each genotype, the median green fluorescence of the infected population was normalized to the median fluorescence of the uninfected population.

N. parisii spores were prepared as previously described [33 (link)]. 0.5 million spores were mixed with OP50-1, M9, and 1,200 synchronized L1 animals, and the mix was top-plated onto 6-cm NGM plates; at least two replicates (two plates) per genotype were set up per infection assay. Three independent infection assays were set up for each timepoint described below. Animals were infected at 25°C for 3 h or 30 h before fixation in either 4% paraformaldehyde or 100% acetone. Fixed worms were incubated at 46°C overnight with FISH probes conjugated to the red Cal Fluor 610 fluorophore that hybridize to N. parisii ribosomal RNA (Biosearch Technologies, Hoddeson, United Kingdom). 3 hpi samples were analyzed for N. parisii sporoplasms using a AxioImager M1 compound microscope (Zeiss). 30 hpi samples were analyzed for N. parisii meronts using a COPAS Biosort machine (Union Biometrica, Holliston, MA) where FISH signal for each worm was normalized to the length of the worm using time-of-flight measurements.